U.S. patent application number 11/602804 was filed with the patent office on 2007-11-22 for multitargeting interfering rnas having two active strands and methods for their design and use.
Invention is credited to Gregory Martin Arndt, Donald John Birkett, Toby Passioura, Michael Poidinger, Laurent Pierre Rivory.
Application Number | 20070269815 11/602804 |
Document ID | / |
Family ID | 38048226 |
Filed Date | 2007-11-22 |
United States Patent
Application |
20070269815 |
Kind Code |
A1 |
Rivory; Laurent Pierre ; et
al. |
November 22, 2007 |
Multitargeting interfering RNAs having two active strands and
methods for their design and use
Abstract
Interfering RNA molecules are now designed and produced with
specificity for multiple binding sequences present in distinct
genetic contexts in one or more pre-selected target RNA molecules
and are used to modulate expression of the target sequences. The
multitargeting interfering RNA molecules have two strands that
target multiple target sites on one or more pre-selected RNA
molecules. Such a multitargeting interfering RNA approach provides
a powerful tool for gene regulation.
Inventors: |
Rivory; Laurent Pierre;
(Springwood, AU) ; Poidinger; Michael; (Rockdale,
AU) ; Birkett; Donald John; (Mosman, AU) ;
Arndt; Gregory Martin; (Malabar, AU) ; Passioura;
Toby; (Surry Hills, AU) |
Correspondence
Address: |
PHILIP S. JOHNSON;JOHNSON & JOHNSON
ONE JOHNSON & JOHNSON PLAZA
NEW BRUNSWICK
NJ
08933-7003
US
|
Family ID: |
38048226 |
Appl. No.: |
11/602804 |
Filed: |
November 21, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60738441 |
Nov 21, 2005 |
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60738640 |
Nov 21, 2005 |
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Current U.S.
Class: |
435/6.18 ;
435/235.1; 435/243; 435/320.1; 435/41; 435/455; 435/468; 435/471;
435/6.1; 514/44A; 536/23.1; 536/24.5; 800/285; 800/295; 800/8 |
Current CPC
Class: |
A61K 31/7115 20130101;
A61K 31/712 20130101; C12N 15/1136 20130101; A61P 11/00 20180101;
C12N 15/1135 20130101; A61P 31/14 20180101; A61K 31/713 20130101;
C12N 2320/50 20130101; C12N 15/1132 20130101; C12N 2310/14
20130101; A61P 31/12 20180101; A61P 43/00 20180101; A61P 31/16
20180101; C12N 15/111 20130101; A61K 31/7105 20130101; A61P 31/18
20180101; C12N 15/1138 20130101; C12N 15/113 20130101 |
Class at
Publication: |
435/006 ;
435/235.1; 435/243; 435/320.1; 435/041; 435/455; 435/468; 435/471;
514/044; 536/024.5; 800/295; 800/008; 536/023.1; 800/285 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C07H 21/04 20060101 C07H021/04; C07H 21/02 20060101
C07H021/02; A01H 5/00 20060101 A01H005/00 |
Claims
1. A multitargeting interfering RNA molecule comprising Formula
(I): 5'-p-XSY-3' 3'-X'S'Y'-p-5' wherein p consists of a terminal
phosphate group that is independently present or absent; wherein S
consists of a first nucleotide sequence of a length of about 5 to
about 20 nucleotides that is completely complementary to a first
portion of a first binding sequence, and S' consists of a second
nucleotide sequence of a length of about 5 to about 20 nucleotides
that is completely complementary to a first portion of a second
binding sequence, wherein said first and second binding sequences
are present in distinct genetic contexts in at least one
pre-selected target RNA molecule, and wherein S and S' are at least
substantially complementary to each other but are not palindromic;
wherein X, X', Y, or Y', is independently absent or consists of a
nucleotide sequence; wherein XSY is at least partially
complementary to the first binding sequence to allow stable
interaction therewith; wherein Y'S'X' is at least partially
complementary to the second binding sequence to allow stable
interaction therewith and is at least partially complementary to
XSY to form a stable duplex therewith.
2. The multitargeting interfering RNA molecule of claim 1, wherein
X, X', Y, or Y', independently consists of one or more
nucleotides.
3. The multitargeting interfering RNA molecule of claim 1, wherein
X consists of a third nucleotide sequence that is at least
partially complementary to a second portion of the first binding
sequence, said second portion is adjacent to and connected with the
3'-end of said first portion of the first binding sequence, and
wherein X' consists of a fourth nucleotide sequence that is
substantially complementary to the third nucleotide sequence.
4. The multitargeting interfering RNA molecule of claim 3, wherein
X and X' are completely complementary to each other.
5. The multitargeting interfering RNA molecule of claim 3, wherein
X is completely complementary to the second portion of the first
binding sequence.
6. The multitargeting interfering RNA molecule of claim 1, wherein
Y' consists of a fifth nucleotide sequence that is at least
partially complementary to a second portion of the second binding
sequence, said second portion is adjacent to and connected with the
3'-end of said first portion of the second binding sequence, and
wherein Y consists of a sixth nucleotide sequence that is
substantially complementary to the fifth nucleotide sequence.
7. The multitargeting interfering RNA molecule of claim 6, wherein
Y and Y' are completely complementary to each other.
8. The multitargeting interfering RNA molecule of claim 6, wherein
Y' is completely complementary to the second portion of the second
binding sequence.
9. The multitargeting interfering RNA molecule of claim 1, wherein
S and S' are completely complementary to each other.
10. The multitargeting interfering RNA molecule of claim 1, wherein
XS is completely complementary to the first portion and the second
portion of the first binding sequence.
11. The multitargeting interfering RNA molecule of claim 1, wherein
Y'S' is completely complementary to the first portion and the
second portion of the second binding sequence.
12. The multitargeting interfering RNA molecule of claim 1, wherein
XSY and Y`S`X' are completely complementary to each other.
13. The multitargeting interfering RNA molecule of claim 1, wherein
S consists of a first nucleotide sequence of a length of about 8 to
about 15 nucleotides.
14. The multitargeting interfering RNA molecule of claim 1, wherein
each of XSY and Y'S'X' is of a length of about 15 to about 29
nucleotides.
15. The multitargeting interfering RNA molecule of claim 1, wherein
each of XSY and Y'S'X' is of a length of about 19 to about 23
nucleotides.
16. The multitargeting interfering RNA molecule of claim 1
comprising one or more terminal overhangs.
17. The multitargeting interfering RNA molecule of claim 16,
wherein the overhang consists of 1 to 5 nucleotides.
18. The multitargeting interfering RNA molecule of claim 1
comprising at least one modified ribonucleotide, universal base,
acyclic nucleotide, abasic nucleotide or non-ribonucleotide.
19. The multitargeting interfering RNA molecule of claim 18
comprising at least one 2'-O-methyl ribosyl substitution or a
locked nucleic acid ribonucleotide.
20. The multitargeting interfering RNA molecule of claim 1, wherein
the first and the second binding sequences are present in distinct
genetic contexts in one pre-selected target RNA molecule.
21. The multitargeting interfering RNA molecule of claim 1, wherein
the first and the second binding sequences are present in distinct
genetic contexts in at least two pre-selected target RNA
molecules.
22. The multitargeting interfering RNA molecule of claim 1, wherein
at least one of the pre-selected target RNA molecules is a
non-coding RNA molecule.
23. The multitargeting interfering RNA molecule of claim 1, wherein
at least one of the pre-selected target RNA molecules is a mRNA
molecule.
24. The multitargeting interfering RNA molecule of claim 1, wherein
at least one of the binding sequences is present in the
3'-untranslated region (3'UTR) of a mRNA molecule.
25. The multitargeting interfering RNA molecule of claim 1, wherein
one or more of the pre-selected target RNA molecules are involved
in a disease or disorder of a biological system.
26. The multitargeting interfering RNA molecule of claim 25,
wherein one or more of the pre-selected target RNA molecules are
involved in a disease or disorder of an animal or a plant.
27. The multitargeting interfering RNA molecule of claim 26,
wherein the animal is selected from the group consisting of a rat,
a mouse, a dog, a cat, a pig, a monkey, and a human.
28. The multitargeting interfering RNA molecule of claim 26,
wherein one or more of the pre-selected target RNA molecules encode
a protein of a class selected from the group consisting of
receptors, cytokines, transcription factors, regulatory proteins,
signaling proteins, cytoskeletal proteins, transporters, enzymes,
hormones, and antigens.
29. The multitargeting interfering RNA molecule of claim 28,
wherein one or more of the pre-selected target RNA molecules encode
a protein selected from the group consisting of ICAM-1, VEGF-A,
MCP-1, IL-8, VEGF-B, IGF-1, Gluc6p, Inppl1, bFGF, PlGF, VEGF-C,
VEGF-D, .beta.-catenin, .kappa.-ras-B, .kappa.-ras-A, EGFR, and TNF
alpha.
30. The multitargeting interfering RNA molecule of claim 29 that
decreases expression of any combination of ICAM-1, VEGF-B, VEGF-C,
VEGF-D, IL-8, bFGF, PlGF, MCP-1 and IGF-1 in an expression
system.
31. The multitargeting interfering RNA molecule of claim 29 that
decreases expression of any combination of ICAM-1, VEGF-A and IGF-1
in an expression system.
32. The multitargeting interfering RNA molecule of claim 29 that
decreases expression of both ICAM-1 and VEGF-A in an expression
system.
33. The multitargeting interfering RNA molecule of claim 29 that
decreases expression of any combination of .beta.-catenin, K-ras,
and EGFR in an expression system.
34. The multitargeting interfering RNA molecule of claim 29 that
decreases expression of both Gluc6p and Inppl1 in an expression
system.
35. The multitargeting interfering RNA molecule of claim 1, wherein
one or more of the pre-selected target RNA molecules encode a viral
RNA.
36. The multitargeting interfering RNA molecule of claim 35,
wherein the virus is selected from the group consisting of a human
immunodeficiency virus (HIV), a hepatitis C virus (HCV), an
influenza virus, a rhinovirus, and a severe acute respiratory
syndrome (SARS) virus.
37. The multitargeting interfering RNA molecule of claim 36,
wherein the virus is a hepatitis C virus (HCV) and a pre-selected
target RNA molecules encodes TNFalpha.
38. The multitargeting interfering RNA molecule of claim 1, wherein
one or more of the pre-selected target RNA molecules comprises one
or more RNA molecules selected from a first biological system.
39. The multitargeting interfering RNA molecule of claim 1, wherein
one or more of the pre-selected target RNA molecules comprises one
or more RNA molecules selected from a second biological system that
is infectious to a first biological system.
40. The multitargeting interfering RNA molecule of claim 1, wherein
the pre-selected target RNA molecules comprise one or more RNA
molecules selected from a first biological system and one or more
pre-selected target RNA molecules selected from a second biological
system that is infectious to the first biological system.
41. The multitargeting interfering RNA molecule of claim 40,
wherein the pre-selected target RNA molecules comprise one or more
RNA molecules selected from an animal or a plant and one or more
RNA molecules selected from a microbe or a virus that is infectious
to the animal or the plant.
42. The multitargeting interfering RNA of claim 41, wherein the
pre-selected target RNA molecules comprises an RNA molecule
encoding a human protein TNFalpha, LEDGF(p75), BAF, CCR5, CXCR4,
furin, NFkB, STAT1.
43. The multitargeting interfering RNA molecule of claim 1, wherein
S consists essentially of a nucleotide sequence selected from the
group consisting of: GUGACAGUCACU (SEQ ID NO: 2), CUGGGCGAGGCAG
(SEQ ID NO: 21), GUGGAUGUGGAG (SEQ ID NO: 22), AGAATCGCAAAACCAGC
(SEQ ID NO: 34), AGAATCGCAAAACCA (SEQ ID NO: 36), CAGGGGAGU (SEQ ID
NO: 46), AGGGCUCCAGGCG (SEQ ID NO: 63) and GCUGGCCGAGGAG. (SEQ ID
NO: 64)
44. The multitargeting interfering RNA molecule of claim 1, wherein
S' consists essentially of a nucleotide sequence selected from the
group consisting of: AGTGACTGTCAC (SEQ ID NO: 1), CUGCCUCGCCCAG
(SEQ ID NO: 19), CUCCACAUCCAC (SEQ ID NO: 20), GCTGGTTTTGCGATTCT
(SEQ ID NO: 33), TGGTTTTGCGATTCT (SEQ ID NO: 35), ACTCCCCTG (SEQ ID
NO: 41), CGCCTGGAGCCCT (SEQ ID NO: 61) and CTCCTCGGCCAGC. (SEQ ID
NO: 62)
45. The multitargeting interfering RNA molecule of claim 1,
consisting essentially of: TABLE-US-00018
5'-CGAGUGACAGUCACUAGCUCC-3' (SEQ ID NO: 3)
3'-UAGCUCACUGUCAGUGAUCGA-5'; (SEQ ID NO: 4)
5'-UCGAGUGACAGUCACUAGCUC-3' (SEQ ID NO: 7)
3'-CUAGCUCACUGUCAGUGAUCG-5'; (SEQ ID NO: 8)
5'-UCGAGUGACAGUCACUAGCUCC-3' (SEQ ID NO: 11)
3'-CUAGCUCACUGUCAGUGAUCGA-5'; (SEQ ID NO: 12)
5'-CGAGUGACAGUCACUAGCUCC-3' (SEQ ID NO: 3)
3'-UAGUUCACUGUCAGUGAUCGA-5'; (SEQ ID NO: 14)
5'-UCGAGUGACAGUCACUAGUUC-3' (SEQ ID NO: 15)
3'-CUAGCUCACUGUCAGUGAUCG-5'; (SEQ ID NO: 8)
5'-CGAGUGACAGUCACUGAUUCC-3' (SEQ ID NO: 16)
3'-CUAGCCACUGUCAGUGAUCGA-5'; (SEQ ID NO: 17)
5'-GAUCGAGUGACAGUCACUAGCUC-3' (SEQ ID NO: 65)
3'-CUAGCUCACUGUCAGUGAUCG-5'; (SEQ ID NO: 8)
5'-CCUCACAGGGGAGUUGUGCCC-3' (SEQ ID NO: 57)
3'-UCGGAGUGUCCCCUCAACACG-5'; (SEQ ID NO: 58) and
5'-CCUCACAGGGGAGUUGUGCUU-3' (SEQ ID NO: 59)
3'-UUGGAGUGUCCCCUCAACACG-5'; (SEQ ID NO: 60)
46. A biological system comprising a multitargeting interfering RNA
molecule comprising Formula (I): 5'-p-XSY-3' 3'-X'S'Y'-p-5' wherein
p consists of a terminal phosphate group that is independently
present or absent; wherein S consists of a first nucleotide
sequence of a length of about 5 to about 20 nucleotides that is
completely complementary to a first portion of a first binding
sequence, and S' consists of a second nucleotide sequence of a
length of about 5 to about 20 nucleotides that is completely
complementary to a first portion of a second binding sequence,
wherein said first and second binding sequences are present in
distinct genetic contexts in at least one pre-selected target RNA
molecule, and wherein S and S' are at least substantially
complementary to each other but are not palindromic; wherein X, X',
Y, or Y', is independently absent or consists of a nucleotide
sequence; wherein XSY is at least partially complementary to the
first binding sequence to allow stable interaction therewith;
wherein Y'S'X' is at least partially complementary to the second
binding sequence to allow stable interaction therewith and is at
least partially complementary to XSY to form a stable duplex
therewith.
47. The biological system of claim 46 being a virus, a microbe, a
cell, a plant, or an animal.
48. A vector comprising a nucleotide sequence that encodes the
multitargeting interfering RNA molecule comprising Formula (I):
5'-p-XSY-3' 3'-X'S'Y'-p-5' wherein p consists of a terminal
phosphate group that is independently present or absent; wherein S
consists of a first nucleotide sequence of a length of about 5 to
about 20 nucleotides that is completely complementary to a first
portion of a first binding sequence, and S' consists of a second
nucleotide sequence of a length of about 5 to about 20 nucleotides
that is completely complementary to a first portion of a second
binding sequence, wherein said first and second binding sequences
are present in distinct genetic contexts in at least one
pre-selected target RNA molecule, and wherein S and S' are at least
substantially complementary to each other but are not palindromic;
wherein X, X', Y, or Y', is independently absent or consists of a
nucleotide sequence; wherein XSY is at least partially
complementary to the first binding sequence to allow stable
interaction therewith; wherein Y'S'X' is at least partially
complementary to the second binding sequence to allow stable
interaction therewith and is at least partially complementary to
XSY to form a stable duplex therewith.
49. The vector of claim 48 being a viral vector.
50. The vector of claim 49 that is derived from a virus selected
from the group consisting of an adeno-associated virus, a
retrovirus, an adenovirus, a lentivirus, and an alphavirus.
51. A cell comprising a vector wherein the vector comprises a
nucleotide sequence that encodes the multitargeting interfering RNA
molecule comprising Formula (I): 5'-p-XSY-3' 3'-X'S'Y'-p-5' wherein
p consists of a terminal phosphate group that is independently
present or absent; wherein S consists of a first nucleotide
sequence of a length of about 5 to about 20 nucleotides that is
completely complementary to a first portion of a first binding
sequence, and S' consists of a second nucleotide sequence of a
length of about 5 to about 20 nucleotides that is completely
complementary to a first portion of a second binding sequence,
wherein said first and second binding sequences are present in
distinct genetic contexts in at least one pre-selected target RNA
molecule, and wherein S and S' are at least substantially
complementary to each other but are not palindromic; wherein X, X',
Y, or Y', is independently absent or consists of a nucleotide
sequence; wherein XSY is at least partially complementary to the
first binding sequence to allow stable interaction therewith;
wherein Y'S'X' is at least partially complementary to the second
binding sequence to allow stable interaction therewith and is at
least partially complementary to XSY to form a stable duplex
therewith.
52. The multitargeting interfering RNA molecule of claim 1 wherein
the molecule is a short hairpin RNA molecule.
53. A vector for a short hairpin RNA molecule wherein the short
hairpin RNA molecule is a multitargeting interfering RNA comprising
Formula (I): 5'-p-XSY-3' 3'-X'S'Y'-p-5' wherein p consists of a
terminal phosphate group that is independently present or absent;
wherein at least one of the terminal phosphate groups is absent and
the strand XSY is linked to the strand Y'S'X' 3' to 5' or 5' to 3'
via a linker wherein S consists of a first nucleotide sequence of a
length of about 5 to about 20 nucleotides that is completely
complementary to a first portion of a first binding sequence, and
S' consists of a second nucleotide sequence of a length of about 5
to about 20 nucleotides that is completely complementary to a first
portion of a second binding sequence, wherein said first and second
binding sequences are present in distinct genetic contexts in at
least one pre-selected target RNA molecule, and wherein S and S'
are at least substantially complementary to each other but are not
palindromic; wherein X, X', Y, or Y', is independently absent or
consists of a nucleotide sequence; wherein XSY is at least
partially complementary to the first binding sequence to allow
stable interaction therewith; wherein Y'S'X' is at least partially
complementary to the second binding sequence to allow stable
interaction therewith and is at least partially complementary to
XSY to form a stable duplex therewith.
54. A cell comprising the vector of claim 53.
55. A pharmaceutical composition comprising a multitargeting
interfering RNA molecule of claim 1 and an acceptable carrier.
56. A pharmaceutical composition comprising a vector of claim 48
and an acceptable carrier.
57. A pharmaceutical composition comprising a vector of claim 53
and an acceptable carrier.
58. A method of inducing RNA interference in a biological system,
comprising the step of introducing a multitargeting interfering RNA
molecule of claim 1 into the biological system wherein the RNA
molecule contacts target RNA and inhibits target RNA activity.
59. A method for designing a multitargeting interfering RNA
molecule, comprising the steps of: a) selecting one or more target
RNA molecules, wherein the modulation in expression of the target
RNA molecules is desired; b) obtaining at least one nucleotide
sequence for each of the target RNA molecules; c) selecting a
length, n, in nucleotides, for a seed sequence, wherein n=about 6
or more; d) obtaining a collection of candidate seeds of the length
n from each nucleotide sequence obtained in step b), wherein a
candidate seed and its complete complement are not palindromic, and
the candidate seed occurs at least once in one or more of the
nucleotide sequences obtained in step b), and its complete
complement occurs at least once in one or more of the nucleotide
sequences obtained in step b); e) determining the genetic context
of each of the candidate seed and its complete complement, by
collecting, for each occurrence of the candidate seed and its
complete complement, a desired amount of the 5' and 3' flanking
sequence; f) selecting a seed of the length n from the group of
candidate seed; g) selecting a first consensus target sequence,
which comprises the seed and a consensus 3'-flanking sequence to
the seed determined from the sequences obtained in step b); h)
selecting a second consensus target sequence, which comprises the
complete complement of the seed and a consensus 3'-flanking
sequence to the complete complement of the seed determined from the
sequences obtained in step b); i) obtaining a first strand
sequence, which comprises the first consensus target sequence
selected in step g) and, adjacent to and connected with the 5'-end
of the first consensus target sequence, a complement of the
consensus 3' flanking sequence of step h); j) obtaining a second
strand sequence which comprises the second consensus target
sequence selected in step h) and, adjacent to and connected with
the 5'-end of the second consensus target sequence, a complement of
the consensus 3' flanking sequence of step g), and; k) designing a
multitargeting interfering RNA molecule comprising a first strand
having the first strand sequence in step i) and a second strand
having the second strand sequence obtained in step j).
60. The method of claim 59 wherein the step of obtaining a
collection of candidate seeds of the length n comprises the steps
of: i) generating a first collection of sequences of the length n
from each of the nucleotide sequences obtained in step b) of claim
59, using a method comprising the steps of: 1) beginning at a
terminus of each of the nucleotide sequence; 2) sequentially
observing the nucleotide sequence using a window size of n; and 3)
stepping along the nucleotide sequence with a step size of 1; ii)
generating a second collection of sequences each of which is
completely complementary to a sequence in the first collection; and
iii) obtaining the collection of candidate seeds of the length n
from the inspection of the first and the second collections of
sequences, wherein a candidate seed and its complete complement are
not palindromic, and each candidate seed and its complete
complement occurs at least once in the nucleotide sequences
obtained in step b) of claim 59.
61. The method of claim 59 wherein the step of obtaining a
collection of candidate seeds of the length n comprises the steps
of: i) obtaining the completely complementary sequence for each
nucleotide sequence obtained in step (b) of claim 59; ii)
generating a first collection of sequences of the length n from
each of the nucleotide sequences obtained in step b) of claim 59
and a second collection of sequences of the length n from each of
the completely complementary sequences obtained in step (i), using
a method comprising the steps of: 1) beginning at a terminus of the
nucleotide sequence of each of the nucleotide sequences obtained in
step b) of claim 59 or each of the completely complementary
sequences obtained in step (i); 2) sequentially observing the
nucleotide sequence using a window size of n; and 3) stepping along
the nucleotide sequence with a step size of 1; and iii) obtaining
the collection of candidate seeds of the length n from the
inspection of the first and the second collections of sequences,
wherein a candidate seed and its complete complement are not
palindromic, and each of the candidate seeds is present in both the
first and the second collections of sequences.
62. The method of claim 59, wherein the step of selecting a group
of candidate seeds comprises the step of discarding any sequence of
the length n that i) is composed of a consecutive string of 5 or
more identical single nucleotides; ii) is composed of only
adenosine and uracil; iii) is predicted to occur with unacceptable
high frequency in the non-target transcriptome of interest; iv) is
predicted to have a propensity to undesirably modulate the
expression or activity of one or more cellular component; v) is any
combination of i) to iv); or vi) is palindromic.
63. The method of claim 59, wherein each of the steps of selecting
a first and a second consensus target sequence comprises the step
of discarding any sequence that i) is composed of only a single
base; ii) is composed of only adenosine and uracil; iii) has a
consecutive string of five or more bases which are cytosine; iv) is
predicted to occur with unacceptable high frequency in the
non-target transcriptome of interest; v) is predicted to have a
propensity to undesirably modulate the expression or activity of
one or more cellular component; or vi) is any combination of i) to
v).
64. The method of claim 59, further comprising the step of
modifying the multitargeting interfering RNA molecule, i) to
improve the incorporation of the first and the second strands of
the multitargeting interfering RNA molecule into the RNA induced
silencing complex (RISC); ii) to increase or decrease the
modulation of the expression of at least one target RNA molecule;
iii) to decrease stress or inflammatory response when the
multitargeting interfering RNA molecule is administered into a
subject; iv) to alter half life in an expression system; or iv) any
combination of i) to iv).
65. The method of claim 59, further comprising repeating the steps
c) to k) of claim 59 with a new value of n.
66. The method of claim 59, further comprising the steps of making
the designed multitargeting interfering RNA molecule and testing it
in an expression system.
67. The method of claim 59, wherein in the step of selecting a
first consensus target sequence, the consensus 3' flanking sequence
to the seed comprises a sequence that is at least partially
identical to the 3' flanking sequence to the seed in at least one
sequence obtained in step b) of claim 59.
68. The method of claim 67, wherein the consensus 3'-flanking
sequence to the seed comprises a sequence that is identical to the
3' flanking sequence to the seed in at least one sequence obtained
in step b) of claim 59.
69. The method of claim 59, wherein in the step of selecting a
second consensus target sequence, the consensus 3' flanking
sequence to the complete complement of the seed comprises a
sequence that is at least partially identical to the 3' flanking
sequence to the complete complement of the seed in at least one
sequence obtained in step b) of claim 59.
70. The method of claim 69, wherein the consensus 3' flanking
sequence to the complete complement of the seed comprises a
sequence that is identical to the 3'-flanking sequence to the seed
in the sequences obtained in step b) of claim 59.
71. The method of claim 59, wherein in the step of obtaining a
first strand sequence, the complement of the consensus 3' flanking
sequence is a complete complement of the consensus 3' flanking
sequence of step h) of claim 59.
72. The method of claim 59, wherein in the step of obtaining a
second strand sequence, the complement of the consensus 3' flanking
sequence is a complete complement of the consensus 3' flanking
sequence of step g) of claim 59.
73. The method of claim 59, wherein in the step of designing a
multitargeting interfering RNA molecule, the first strand and the
second strand are completely complementary to each other, excepting
the overhangs if present.
74. The method of claim 59, wherein in the step of designing a
multitargeting interfering RNA molecule, the first strand and the
second strand are incompletely complementary to each other.
75. A method of treating a subject, comprising the step of
administering to said subject a therapeutically effective amount of
a pharmaceutical composition comprising a multitargeting
interfering RNA molecule of claim 1.
76. The method of claim 75, further comprising administering to
said subject a therapeutically effective amount of one or more
additional therapeutic agents.
77. A method of inhibiting the onset of a disease or condition in a
subject, comprising administering to said subject a
prophylactically effective amount of a pharmaceutical composition
comprising at least one multitargeting interfering RNA molecule of
claim 1.
78. A process for making a pharmaceutical composition comprising
mixing a multitargeting interfering RNA molecule of claim 1 and a
pharmaceutically acceptable carrier.
79. A method of introducing a multitargeting interfering RNA
molecule comprising Formula (I) into a cell comprising the steps
of: i) generating a multitargeting interfering RNA molecule
comprising Formula (I) 5'-p-XSY-3' 3'-X'S'Y'-p-5' wherein p
consists of a terminal phosphate group that is independently
present or absent; wherein S consists of a first nucleotide
sequence of a length of about 5 to about 20 nucleotides that is
completely complementary to a first portion of a first binding
sequence, and S' consists of a second nucleotide sequence of a
length of about 5 to about 20 nucleotides that is completely
complementary to a first portion of a second binding sequence,
wherein said first and second binding sequences are present in
distinct genetic contexts in at least one pre-selected target RNA
molecule, and wherein S and S' are at least substantially
complementary to each other but are not palindromic; wherein X, X',
Y, or Y', is independently absent or consists of a nucleotide
sequence; wherein XSY is at least partially complementary to the
first binding sequence to allow stable interaction therewith;
wherein Y'S'X' is at least partially complementary to the second
binding sequence to allow stable interaction therewith and is at
least partially complementary to XSY to form a stable duplex
therewith; and ii) contacting the multitargeting interfering RNA
molecule with a cell.
80. The method of claim 79 wherein the multitargeting interfering
RNA is encoded by DNA.
81. The method of claim 79 wherein the RNA is encoded by a DNA or
RNA vector
82. The method of claim 79 wherein the contacting step further
comprises the step of introducing the RNA molecule or an RNA
molecule encoded by a DNA or RNA vector into the cell.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application Nos. 60/738,441 filed Nov. 21, 2005 and
60/738,640 filed Nov. 21, 2005, respectively, which are
incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention concerns methods and reagents useful
in modulating gene expression. Particularly, the invention relates
to modulating gene expression using one multitargeting interfering
RNA molecule having two strands each of which targets one or more
sites on one or more pre-selected RNA molecules.
BACKGROUND OF THE INVENTION
[0003] It is now known that single and double-stranded RNA can
modulate expression of or modify processing of target RNA molecules
by a number of mechanisms. Some such mechanisms tolerate variation
in the amount of sequence complementarity required between the
modulatory (or interfering) RNA and the target RNA. Certain
microRNAs can translationally repress target mRNA having as little
as 6 nucleotides of complementarity with the microRNA. The
development of RNA interference agents, for example, using
double-stranded RNA to repress expression of disease-related genes
is currently an area of intense research activity.
[0004] Double-stranded RNA of 19-23 bases in length is recognized
by an RNA interference silencing complex (RISC) into which an
effector strand (or "guide strand") of the RNA is loaded. This
guide strand acts as a template for the recognition and destruction
of highly complementary sequences present in the transcriptome.
Alternatively, through the recognition and binding of RNA sequences
of lower complementarity, interfering RNAs may induce translational
repression without mRNA degradation. Such translational repression
appears to be a mechanism of action of endogenous microRNAs, a
group of short non-coding RNAs involved in differentiation and
development.
[0005] Efforts at developing interfering RNAs for therapeutically
applications thus far have focused on producing specific
double-stranded RNAs, each with complete complementarity to a
particular target transcript. Such double-stranded RNAs (dsRNAs)
are potentially effective where a single suitable target can be
identified. However, dsRNAs, particularly those designed against
one target, may have at least two categories of off-target side
effects that need to be avoided or minimized. Undesirable side
effects can arise through the triggering of innate immune response
pathways (e.g. Toll-like Receptors 3, 7, and 8, and the so-called
interferon response) and through inadvertent inhibition of protein
expression from related or unrelated transcripts (either by RNA
degradation, translational repression or other mechanisms).
Inadvertent side-effects can be obtained when the passenger strand
of a duplex is loaded and generates suppression of RNA species
distinct from those targeted by the putative guide strand. Loading
bias is well understood and most design processes only select
sequences for a RNAi duplex from which only the intended guide
strand will be loaded. Thus, some bioinformatic and/or experimental
approaches have been developed to try to minimize off-target
effects. Algorithms for in silico hybridization are known, and
others have been developed for predicting target accessibility and
loading bias in an effort to eliminate or minimize side-effects
while maintaining effectiveness.
[0006] Several double-stranded RNA molecules for potentially
treating human diseases of viral and non-viral origin are in
various stages of development. The diseases include Age-related
Macular Degeneration, Amyotrophic Lateral Sclerosis (ALS), and
Respiratory Syncytial Virus (RSV) infection. These RNA molecules,
however, only target a single site in an RNA sequence. Although RNA
interference may be useful and potent in obtaining knock-down of
specific gene products, many diseases involve complex interactions
between ontologically-unrelated gene products. Thus, the use of
single-gene targeting approaches may not succeed except where a
single or dominant pathophysiologic pathway can be identified and
interrupted.
[0007] In fact, many putative targets can be identified for most
diseases. Attempts to confirm that inhibiting single targets in
isolation is therapeutically valuable have been disappointing.
Indeed, obtaining therapeutic effectiveness is proving to be
extremely challenging, probably because of multiple levels of
redundancy in most signaling pathways. For example, many disorders,
such as cancer, type 2 diabetes, and atherosclerosis, feature
multiple biochemical abnormalities. In addition, some putative
targets may be subject to enhanced mutation rates, thereby negating
the effects of interfering RNAs on any such target.
[0008] For example, therapeutic approaches to viral infections
continue to be major challenges in agriculture, as well as in
animal and human health. The nature of the replication of viruses
makes them highly plastic, "moving targets"
therapeutically--capable of altering structure, infectivity, and
host profile. The recent emergence of viruses such as Severe Acute
Respiratory Syndrome ("SARS") and Avian Influenza Virus ("bird
flu") exemplify these challenges. Even well-described viruses such
as those involved in Acquired Immunodeficiency Syndrome or AIDS
(e.g. Human Immunodeficiency Viruses, HIV-1 and HIV-2), continue to
defy efforts at treatment and vaccination because of on-going viral
mutation and evolution.
[0009] Furthermore, although nucleic acid therapeutics such as
interfering RNAs are candidates for viral therapy, in part because
modern rapid gene sequencing techniques allow viral genome
sequences to be determined even before any encoded functions can be
assessed, the error-prone replication of viruses, particularly RNA
viruses, means that substantial genomic diversity can arise rapidly
in an infected population. Thus far, strategies for the development
of nucleic acid therapeutics have largely centered on the targeting
of highly-conserved regions of the viral genome. It is unclear
whether these constructs are efficient at treating viral infection
or preventing emergence of resistant viral clones.
[0010] Therapeutic approaches that involve the design and use of
one interfering RNA for control of several key "drivers" of the
disease are thus desirable. Therefore, there is a need for
interfering RNAs which can modulate multiple RNAs or target
multiple sites within an RNA. Methods for the design and for making
such therapeutic multi-targeting interfering RNAs are also needed.
Antiviral interfering RNAs that can be developed rapidly upon the
isolation and identification of new viral pathogens and that can be
used to help slow, or even prevent, the emergence of new, resistant
isotypes are also needed. Finally, it would be useful to have such
RNAs wherein each of the two strands of a synthetic duplex
independently targets at least one of the multiple target RNAs.
SUMMARY OF THE INVENTION
[0011] Interfering RNA molecules are now designed and produced with
specificity for multiple binding sequences present in distinct
genetic contexts in one or more pre-selected target RNA molecules
and are used to modulate expression of the target sequences.
[0012] In a first embodiment, the present invention relates to a
multitargeting interfering RNA molecule comprising Formula (I):
5'-p-XSY-3' 3'-X'S'Y'-p-5' wherein p consists of a terminal
phosphate group that is independently present or absent; wherein S
consists of a first nucleotide sequence of a length of about 5 to
about 20 nucleotides that is completely complementary to a first
portion of a first binding sequence, and S' consists of a second
nucleotide sequence of a length of about 5 to about 20 nucleotides
that is completely complementary to a first portion of a second
binding sequence, wherein said first and second binding sequences
are present in distinct genetic contexts in at least one
pre-selected target RNA molecule, and wherein S and S' are at least
substantially complementary to each other but are not palindromic;
and further wherein X, X', Y, or Y', is independently absent or
consists of a nucleotide sequence; wherein XSY is at least
partially complementary to the first binding sequence to allow
stable interaction therewith; and wherein Y'S'X' is at least
partially complementary to the second binding sequence to allow
stable interaction therewith and is at least partially
complementary to XSY to form a stable duplex therewith.
[0013] In one version of this preferred embodiment, X, X', Y, or
Y', independently consists of one or more nucleotides and in
another aspect of this embodiment X consists of a third nucleotide
sequence that is at least partially complementary to a second
portion of the first binding sequence, where the second portion is
adjacent to and connected with the 3'-end of said first portion of
the first binding sequence, and where X' consists of a fourth
nucleotide sequence that is substantially complementary to the
third nucleotide sequence. Preferably in this aspect X and X' are
completely complementary to each other. More preferably, X is
completely complementary to the second portion of the first binding
sequence.
[0014] In another aspect of this first embodiment, Y' is designed
to consist of a fifth nucleotide sequence that is at least
partially complementary to a second portion of the second binding
sequence and the second portion is adjacent to and connected with
the 3'-end of said first portion of the second binding sequence. In
this aspect Y consists of a sixth nucleotide sequence that is
substantially complementary to the fifth nucleotide sequence.
Preferably Y and Y' are completely complementary to each other or
alternatively Y' is completely complementary to the second portion
of the second binding sequence.
[0015] In yet other aspects of this first preferred embodiment, S
and S' are completely complementary to each other. Alternatively XS
is completely complementary to the first portion and the second
portion of the first binding sequence. It is also contemplated that
Y'S' is completely complementary to the first portion and the
second portion of the second binding sequence. Further, XSY and
Y'S'X' can be completely complementary to each other. Optionally,
in aspects of this invention, S consists of a first nucleotide
sequence of a length of about 8 to about 15 nucleotides and XSY and
Y'S'X' preferably include lengths of about 15 to about 29
nucleotides. Also preferably, each of XSY and Y'S'X' are of a
length of about 19 to about 23 nucleotides. In some aspects of this
embodiment, the multitargeting interfering RNA molecule comprises
one or more terminal overhangs and preferably these overhangs
consists of 1 to 5 nucleotides. In other preferred aspects of this
embodiment, the multitargeting interfering RNA molecule comprises
at least one modified ribonucleotide, universal base, acyclic
nucleotide, abasic nucleotide or non-ribonucleotide and more
preferably, the multitargeting interfering RNA molecule comprises
at least one 2'-O-methyl ribosyl substitution or a locked nucleic
acid ribonucleotide.
[0016] In yet a further aspect of this first embodiment, the first
and the second binding sequences of the multitargeting interfering
RNA molecule are present in distinct genetic contexts in one
pre-selected target RNA molecule or alternatively, the first and
the second binding sequences are present in distinct genetic
contexts in at least two pre-selected target RNA molecules.
Preferably at least one of the pre-selected target RNA molecules is
a non-coding RNA molecule. Also preferably, at least one of the
pre-selected target RNA molecules is a messenger RNA (mRNA). In a
further preferable embodiment at least one of the binding sequences
is present in the 3'-untranslated region (3'UTR) of a mRNA
molecule. Preferably the pre-selected target RNA molecules are
involved in a disease or disorder of a biological system and the
disease or disorder preferably that of an animal or a plant.
Preferred animals include, but are not limited to rat, a mouse, a
dog, a cat, a pig, a monkey, and a human. Further the pre-selected
target RNA molecules encode a protein of a class selected from the
group consisting of receptors, cytokines, transcription factors,
regulatory proteins, signaling proteins, cytoskeletal proteins,
transporters, enzymes, hormones, and antigens. Preferred proteins
include those selected from the group consisting of ICAM-1, VEGF-A,
MCP-1, IL-8, VEGF-B, IGF-1, Gluc6p, Inppl1, bFGF, PlGF, VEGF-C,
VEGF-D, .beta.-catenin, .kappa.-ras-B, .kappa.-ras-A, EGFR, and TNF
alpha and preferably the multitargeting interfering RNA molecule
decreases expression of any combination of ICAM-1, VEGF-B, VEGF-C,
VEGF-D, IL-8, bFGF, PlGF, MCP-1 and IGF-1 in an expression system.
Also preferably the multitargeting interfering RNA molecule
decreases expression of any combination of .beta.-catenin,
.kappa.-ras, and EGFR in an expression system or decreases
expression of both Gluc6p and Inppl1 in an expression system.
Alternatively, the multitargeting interfering RNA targets viral
RNA. Preferred viral targets include human immunodeficiency virus
(HIV), a hepatitis C virus (HCV), an influenza virus, a rhinovirus,
and a severe acute respiratory syndrome (SARS) virus. As one
example, the multitargeting interfering RNA molecule targets
hepatitis C virus (HCV) and an RNA molecule encoding TNFalpha.
[0017] In still further aspects of the present embodiment, one or
more of the pre-selected target RNA molecules preferably comprises
one or more RNA molecules selected from a first biological system.
Alternatively, one or more of the pre-selected target RNA molecules
comprises one or more RNA molecules selected from a second
biological system that is infectious to a first biological system.
In another aspect, the pre-selected target RNA molecules comprise
one or more RNA molecules selected from a first biological system
and one or more pre-selected target RNA molecules selected from a
second biological system that is infectious to the first biological
system. Preferably the pre-selected target RNA molecules comprise
one or more RNA molecules selected from an animal or a plant and
one or more RNA molecules selected from a microbe or a virus that
is infectious to the animal or the plant. The pre-selected target
RNA molecules preferably comprises an RNA molecule encoding a human
protein TNFalpha, LEDGF(p75), BAF, CCR5, CXCR4, furin, NFkB,
STAT1.
[0018] As examples of the multitargeting interfering RNA molecules
of this invention, S preferably consists essentially of a
nucleotide sequence selected from the group consisting of:
GUGACAGUCACU (SEQ ID NO: 2), CUGGGCGAGGCAG (SEQ ID NO: 21),
GUGGAUGUGGAG (SEQ ID NO: 22), AGAATCGCAAAACCAGC (SEQ ID NO: 34),
AGAATCGCAAAACCA (SEQ ID NO: 36), CAGGGGAGU (SEQ ID NO: 46),
AGGGCUCCAGGCG (SEQ ID NO: 63) and GCUGGCCGAGGAG. (SEQ ID NO: 64).
In further examples, S' consists essentially of a nucleotide
sequence selected from the group consisting of: AGTGACTGTCAC (SEQ
ID NO: 1), CUGCCUCGCCCAG (SEQ ID NO: 19), CUCCACAUCCAC (SEQ ID NO:
20), GCTGGTTTTGCGATTCT (SEQ ID NO: 33), TGGTTTTGCGATTCT (SEQ ID NO:
35), ACTCCCCTG (SEQ ID NO: 41), CGCCTGGAGCCCT (SEQ ID NO: 61) and
CTCCTCGGCCAGC. (SEQ ID NO: 62).
[0019] In yet other embodiments, the multitargeting interfering RNA
molecules consist essentially of: TABLE-US-00001
5'-CGAGUGACAGUCACUAGCUCC-3' (SEQ ID NO: 3)
3'-UAGCUCACUGUCAGUGAUCGA-5'; (SEQ ID NO: 4)
5'-UCGAGUGACAGUCACUAGCUC-3' (SEQ ID NO: 7)
3'-CUAGCUCACUGUCAGUGAUCG-5'; (SEQ ID NO: 8)
5'-UCGAGUGACAGUCACUAGCUCC-3' (SEQ ID NO: 11)
3'-CUAGCUCACUGUCAGUGAUCGA-5'; (SEQ ID NO: 12)
5'-CGAGUGACAGUCACUAGCUCC-3' (SEQ ID NO: 3)
3'-UAGUUCACUGUCAGUGAUCGA-5'; (SEQ ID NO: 14)
5'-UCGAGUGACAGUCACUAGUUC-3' (SEQ ID NO: 15)
3'-CUAGCUCACUGUCAGUGAUCG-5'; (SEQ ID NO: 8)
5'-CGAGUGACAGUCACUGAUUCC-3' (SEQ ID NO: 16)
3'-CUAGCCACUGUCAGUGAUCGA-5'; (SEQ ID NO: 17)
5'-GAUCGAGUGACAGUCACUAGCUC-3' (SEQ ID NO: 65)
3'-CUAGCUCACUGUCAGUGAUCG-5'; (SEQ ID NO: 8)
5'CCUCACAGGGGAGUUGUGCCC-3' (SEQ ID NO: 57)
3'-UCGGAGUGUCCCCUCAACACG-5'; (SEQ ID NO: 58) and
5'-CCUCACAGGGGAGUUGUGCUU-3' (SEQ ID NO: 59)
3'-UUGGAGUGUCCCCUCAACACG-5' (SEQ ID NO: 60)
[0020] In another embodiment of the present invention the invention
relates to a biological system comprising a multitargeting
interfering RNA molecule comprising Formula (I): 5'-p-XSY-3'
3'-X'S'Y'-p-5' wherein p consists of a terminal phosphate group
that is independently present or absent; wherein S consists of a
first nucleotide sequence of a length of about 5 to about 20
nucleotides that is completely complementary to a first portion of
a first binding sequence, and S' consists of a second nucleotide
sequence of a length of about 5 to about 20 nucleotides that is
completely complementary to a first portion of a second binding
sequence, wherein said first and second binding sequences are
present in distinct genetic contexts in at least one pre-selected
target RNA molecule, and wherein S and S' are at least
substantially complementary to each other but are not palindromic;
and further wherein X, X', Y, or Y', is independently absent or
consists of a nucleotide sequence; wherein XSY is at least
partially complementary to the first binding sequence to allow
stable interaction therewith; and wherein Y'S'X' is at least
partially complementary to the second binding sequence to allow
stable interaction therewith and is at least partially
complementary to XSY to form a stable duplex therewith. In the
present invention, preferred biological systems include virus,
microbes, cells, plants, or animals.
[0021] The invention further relates to vectors comprising
nucleotide sequences encoding the multitargeting interfering RNA
molecules of this invention. Preferred vectors include viral
vectors and preferred vectors are those selected from the group
consisting of an adeno-associated virus, a retrovirus, an
adenovirus, a lentivirus, and an alphavirus. Cells comprising these
vectors are also contemplated in this invention. Where the
multitargeting interfering RNA molecule is a short hairpin RNA
molecule, vectors capable of encoding these short hairpin RNA
molecule and those cells containing those vectors or the short
hairpin RNA molecules of this invention are also contemplated.
[0022] The invention further relates to pharmaceutical compositions
comprising the multitargeting interfering RNA molecules of this
invention together with an acceptable carrier. Other pharmaceutical
compositions include the vectors of this invention together with
acceptable carriers.
[0023] In yet another embodiment of the present invention, the
invention relates to a method of inducing RNA interference in a
biological system, such as virus, microbes, cells, plants, or
animals. These methods include the steps of introducing the
multitargeting interfering RNA molecules of the present invention
into those biological systems.
[0024] Further embodiments of this invention include methods for
designing multitargeting interfering RNA molecule, comprising the
steps of: a) selecting one or more target RNA molecules, wherein
the modulation in expression of the target RNA molecules is
desired; b) obtaining at least one nucleotide sequence for each of
the target RNA molecules; c) selecting a length, n, in nucleotides,
for a seed sequence, wherein n=about 6 or more; d) obtaining a
collection of candidate seeds of the length n from each nucleotide
sequence obtained in step b), wherein a candidate seed and its
complete complement are not palindromic, and the candidate seed
occurs at least once in one or more of the nucleotide sequences
obtained in step b), and its complete complement occurs at least
once in one or more of the nucleotide sequences obtained in step
b); e) determining the genetic context of each of the candidate
seeds and its complete complement, by collecting, for each
occurrence of the candidate seed and its complete complement, a
desired amount of the 5' and 3' flanking sequence; f) selecting a
seed of the length n from the group of candidate seeds; g)
selecting a first consensus target sequence, which comprises the
seed and a consensus 3'-flanking sequence to the seed determined
from the sequences obtained in step b); h) selecting a second
consensus target sequence, which comprises the complete complement
of the seed and a consensus 3'-flanking sequence to the complete
complement of the seed determined from the sequences obtained in
step b); i) obtaining a first strand sequence, which comprises the
first consensus target sequence selected in step g) and, adjacent
to and connected with the 5'-end of the first consensus target
sequence, a complement of the consensus 3' flanking sequence of
step h); j) obtaining a second strand sequence which comprises the
second consensus target sequence selected in step h) and, adjacent
to and connected with the 5'-end of the second consensus target
sequence, a complement of the consensus 3' flanking sequence of
step g), and; k) designing a multitargeting interfering RNA
molecule comprising a first strand having the first strand sequence
in step i) and a second strand having the second strand sequence
obtained in step j).
[0025] In a preferred aspect of this embodiment, the invention
further comprises the step of obtaining a collection of candidate
seeds of the length n, the steps of: i) generating a first
collection of sequences of the length n from each of the nucleotide
sequences obtained in step b) above using a method comprising the
steps of: 1) beginning at a terminus of each of the nucleotide
sequence; 2) sequentially observing the nucleotide sequence using a
window size of n; and 3) stepping along the nucleotide sequence
with a step size of 1; ii) generating a second collection of
sequences each of which is completely complementary to a sequence
in the first collection; and iii) obtaining the collection of
candidate seeds of the length n from the inspection of the first
and the second collections of sequences, wherein a candidate seed
and its complete complement are not palindromic, and each candidate
seed and its complete complement occurs at least once in the
nucleotide sequences obtained in step b) of the method provided
above.
[0026] In another preferred aspect of this designing embodiment
wherein the step of obtaining a collection of candidate seeds of
the length n comprises the steps of: i) obtaining the completely
complementary sequence for each nucleotide sequence obtained in
step (b) of this designing method; ii) generating a first
collection of sequences of the length n from each of the nucleotide
sequences obtained in step b) and a second collection of sequences
of the length n from each of the completely complementary sequences
obtained in the present method, wherein the generating step
comprises: 1) beginning at a terminus of the nucleotide sequence of
each of the nucleotide sequences obtained in step b) above or each
of the completely complementary sequences obtained in this aspect
of the invention; 2) sequentially observing the nucleotide sequence
using a window size of n; and 3) stepping along the nucleotide
sequence with a step size of 1; and wherein following the
generating step of this aspect the method further comprises iii)
obtaining the collection of candidate seeds of the length n from
the inspection of the first and the second collections of
sequences, wherein a candidate seed and its complete complement are
not palindromic, and each of the candidate seeds is present in both
the first and the second collection of sequences.
[0027] In another preferred aspect of this embodiment, the step of
selecting a group of candidate seeds comprises the step of
discarding any sequence of the length n that: i) is composed of a
consecutive string of 5 or more identical single nucleotides; ii)
is composed of only adenosine and uracil; iii) is predicted to
occur with unacceptable high frequency in the non-target
transcriptome of interest; iv) is predicted to have a propensity to
undesirably modulate the expression or activity of one or more
cellular component; v) is any combination of i) to iv); or vi) is
palindromic. Preferably, each of the steps of selecting a first and
a second consensus target sequence comprises the step of discarding
any sequence that; i) is composed of only a single base; ii) is
composed of only adenosine and uracil; iii) has a consecutive
string of five or more bases which are cytosine; iv) is predicted
to occur with unacceptable high frequency in the non-target
transcriptome of interest; v) is predicted to have a propensity to
undesirably modulate the expression or activity of one or more
cellular component; or vi) is any combination of i) to v).
[0028] The designing methods of this invention may further comprise
the step of modifying the multitargeting interfering RNA molecule,
i) to improve the incorporation of the first and the second strands
of the multitargeting interfering RNA molecule into the RNA induced
silencing complex (RISC); ii) to increase or decrease the
modulation of the expression of at least one target RNA molecule;
iii) to decrease stress or inflammatory response when the
multitargeting interfering RNA molecule is administered into a
subject; iv) to alter half life in an expression system; or v) any
combination of i) to iv).
[0029] The designing methods of this invention preferably further
comprise the steps of making the designed multitargeting
interfering RNA molecule and testing it in a suitable expression
system. Additionally the step of selecting a first consensus target
sequence can further comprises designing the consensus target
sequence where the consensus 3' flanking sequence to the seed
comprises a sequence that is at least partially identical to the 3'
flanking sequence to the seed in at least one sequence obtained in
step b) of the designing steps of this invention. Alternatively,
the consensus 3'-flanking sequence to the seed can comprise a
sequence that is identical to the 3' flanking sequence to the seed
in at least one sequence obtained in step b) of the designing
methods of this invention. Further, in the step of selecting a
second consensus target sequence, in one aspect, the consensus 3'
flanking sequence to the complete complement of the seed comprises
a sequence that is at least partially identical to the 3' flanking
sequence to the complete complement of the seed in at least one
sequence obtained in step b). In other embodiments the consensus 3'
flanking sequence to the complete complement of the seed comprises
a sequence that is identical to the 3'-flanking sequence to the
seed in the sequences obtained in step b). In yet other aspects
related to this designing method, the step of obtaining a first
strand sequence, the complement of the consensus 3' flanking
sequence is a complete complement of the consensus 3' flanking
sequence of step h) of the designing method. Or, also preferably,
in the step of obtaining a second strand sequence, the complement
of the consensus 3' flanking sequence is a complete complement of
the consensus 3' flanking sequence of step g). In a further aspect,
in the step of designing a multitargeting interfering RNA molecule,
the first strand and the second strand are completely complementary
to each other, excepting the overhangs if present or in another
aspect in the step of designing a multitargeting interfering RNA
molecule, the first strand and the second strand are incompletely
complementary to each other.
[0030] In another embodiment of this invention, the invention
relates to a method of treating a subject, comprising the step of
administering to said subject a therapeutically effective amount of
a pharmaceutical composition comprising a multitargeting
interfering RNA molecule of this invention. In a preferred aspect
of this invention, the method further comprises administering to
said subject a therapeutically effective amount of one or more
additional therapeutic agents.
[0031] In yet another embodiment of this invention, the invention
relates to a method of inhibiting the onset of a disease or
condition in a subject, comprising administering to said subject a
prophylactically effective amount of a pharmaceutical composition
comprising at least one multitargeting interfering RNA molecules of
this invention. Other embodiments include processes for making a
pharmaceutical composition comprising mixing a multitargeting
interfering RNA molecules of this invention and a pharmaceutically
acceptable carrier.
[0032] Other aspects of the invention include methods of treating
and methods of inhibiting the onset of a disease or disorder using
a multitargeting interfering RNA of the invention, and methods of
making a pharmaceutical composition comprising a multitargeting
interfering RNA of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1: Multitargeting of VEGF-A and ICAM-1 using both
strands of a CODEMIR duplex. A 12 nt seed region was identified by
analyzing the two target transcripts. Various permutations of
positioning the CODEMIR around the seed were investigated and the
resulting sequences are listed in Table 1-1. A: untransfected
cells; B: irrelevant siRNA control; C: ICAM-1 and VEGF-specific
siRNAs; D: CODEMIR-16; E: CODEMIR-17; F: CODEMIR-26; G: CODEMIR-27;
H: CODEMIR-28 and I: CODEMIR-36. The activity of these CODEMIRs
against ICAM-1 (open bars) and VEGF-A (closed bars) was determined
using RPE cells. CODEMIR-27 and -28 correspond to the duplexes of
CODEMIR-16 and -17, respectively, excepting the introduction of
wobble base-pairs into the extremities of the duplexes to adjust
the loading bias. CODEMIR-36 is an example of an incompletely
complementary duplex formed with guide strands that are fully
complementary to the regions of VEGF-A and ICAM-1 mRNA targeted by
CODEMIR16 and CODEMIR17.
[0034] FIG. 2 Panel A: Further exemplification of multitargeting
using both strands of a CODEMIR duplex in which the CODEMIR duplex
strands may be completely complementary to each other. Any
overhangs present will be without complementary base pairing. Panel
B: An example of a CODEMIR showing incomplete complementarity
between the two active strands of the CODEMIR. Such incomplete
complementarity, can derive, for example, by virtue of each strand
being completely complementary or almost completely complementary
to its respective target.
[0035] FIG. 3. Effect of a single blunt-end on VEGF and ICAM
suppressive activity of CODEMIR targeting these two proteins. A:
untransfected cells; B: mock transfected; C: Irrelevant control
siRNA; D: CODEMIR-17 and E: CODEMIR-103. ARPE-19 cells were
transfected with 40 nM duplex RNA and VEGF (closed bars) or ICAM
(open bars) expression was assayed 48 hours post-transfection. Each
bar represents the mean of triplicate samples. Error bars indicate
standard deviation of the mean.
DETAILED DESCRIPTION
[0036] Various publications, articles and patents are cited or
described in the background and throughout the specification; each
of these references is herein incorporated by reference in its
entirety. Discussion of documents, acts, materials, devices,
articles or the like which has been included in the present
specification is for the purpose of providing context for the
present invention. Such discussion is not an admission that any or
all of these matters form part of the prior art with respect to any
inventions disclosed or claimed.
[0037] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which this invention pertains. In this
invention, certain terms are used frequently, which shall have the
meanings as set forth as follows. These terms may also be explained
in greater detail later in the specification.
[0038] The following are abbreviations that are at times used in
this specification:
[0039] bp=base pair
[0040] cDNA=complementary DNA
[0041] CODEMIR=COmputationally-DEsigned, Multi-targeting
Interfering RNAs
[0042] kb=kilobase; 1000 base pairs
[0043] kDa=kilodalton; 1000 dalton
[0044] miRNA=microRNA
[0045] ncRNA=non-coding RNA
[0046] nt=nucleotide
[0047] PAGE=polyacrylamide gel electrophoresis.
[0048] PCR=polymerase chain reaction
[0049] RISC=RNA interference silencing complex
[0050] RNAi=RNA interference
[0051] SDS=sodium dodecyl sulfate
[0052] siRNA=short interfering RNA
[0053] shRNA=short hairpin RNA
[0054] SNPs=single nucleotide polymorphisms
[0055] UTR=untranslated region
[0056] VIROMIR=multitargeting interfering RNA preferentially
targeted to viral targets
[0057] It must be noted that as used herein and in the appended
claims, the singular forms "a," "an," and "the" include plural
reference unless the context clearly dictates otherwise. Thus, for
example, a reference to "a cell" is a reference to one or more
cells and includes equivalents thereof known to those skilled in
the art and so forth.
[0058] An "activity", a "biological activity", or a "functional
activity" of a polypeptide or nucleic acid refers to an activity
exerted by a polypeptide or nucleic acid molecule as determined in
vivo or in vitro, according to standard techniques. Such activities
can be a direct activity, such as the RNA interfering activity of
an iRNA on a target RNA molecule, or an indirect activity, such as
a cellular signaling activity mediated by the RNA interfering
activity of an iRNA.
[0059] "Biological system" means, material, in a purified or
unpurified form, from biological sources, including but not limited
to human, animal, plant, insect, microbial, viral or other sources,
wherein the system comprises the components required for biologic
activity (e.g., inhibition of gene expression). The term
"biological system" includes, for example, a cell, a virus, a
microbe, an organism, an animal, or a plant.
[0060] A "cell" means an autonomous self-replicating unit that may
constitute an organism (in the case of unicellular organisms) or is
a sub unit of multicellular organisms in which individual cells may
be specialized and/or differentiated for particular functions. A
cell can be prokaryotic or eukaryotic, including bacterial cells
such as E. coli, fungal cells such as yeast, bird cell, mammalian
cells such as cell lines of human, bovine, porcine, monkey, sheep,
apes, swine, dog, cat, and rodent origin, and insect cells such as
Drosophila and silkworm derived cell lines, or plant cells. The
cell can be of somatic or germ line origin, totipotent or hybrid,
dividing or non-dividing. The cell can also be derived from or can
comprise a gamete or embryo, a stem cell, or a fully differentiated
cell. It is further understood that the term "cell" refers not only
to the particular subject cell, but also to the progeny or
potential progeny of such a cell. Because certain modifications can
occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term as used herein.
[0061] The term "complementary" or "complementarity" as used herein
with respect to polynucleotides or oligonucleotides (which terms
are used interchangeably herein) refers to a measure of the ability
of individual strands of such poly- or oligonucleotides to
associate with each other. Two major fundamental interactions in
RNA are stacking and hydrogen bonding. Both contribute to
free-energy changes for associations of oligoribonucleotides. The
RNA-RNA interactions include the standard Watson-Crick pairing (A
opposite U or T, and G opposite C) and the non-Watson-Crick pairing
(including but not limited to the interaction through the Hoogsteen
edge and/or sugar edge) (see e.g., Leontis et al., 2002, Nucleic
Acids Research, 30: 3497-3531). A sequence that is complementary to
another sequence is also referred to as the complement of the
other.
[0062] The degree of complementarity between nucleic acid strands
has significant effects on the efficiency and strength of the
association between the nucleic acid strands. "Complementarity"
between two nucleic acid sequences corresponds to free-energy
changes for helix formation. Thus, determination of binding free
energies for nucleic acid molecules is useful for predicting the
three-dimensional structures of RNAs and for interpreting RNA-RNA
associations. e.g., RNAi activity or inhibition of gene expression
or formation of double stranded oligonucleotides. Such
determination can be made using methods known in the art (see,
e.g., Turner et al., 1987, Cold Spring Harb Symp Quant Biol.
52:123-33; Frier et al., 1986, Proc. Nat. Acad. Sci. USA
83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc.
109:3783-3785).
[0063] As the skilled artisan will appreciate, complementarity,
where present, can be partial, for example where at least one or
more nucleic acid bases between strands can pair according to the
canonical base pairing rules. For example, the sequences
5'-CTGACAATCG-3',5'-CGAAAGTCAG-3' are partially complementary (also
referred to herein as "incompletely complementary") to each other.
"Partial complementarity" or "partially complementary" as used
herein indicates that only a percentage of the contiguous residues
of a nucleic acid sequence can form Watson-Crick base pairing with
the same number of contiguous residues in a second nucleic acid
sequence in an anti-parallel fashion. For example, 5, 6, 7, 8, 9,
or 10 nucleotides out of a total of 10 nucleotides in the first
oligonucleotide forming Watson-Crick base pairing with a second
nucleic acid sequence having 10 nucleotides represents 50%, 60%,
70%, 80%, 90%, and 100% complementarity respectively.
[0064] Complementarity can also be total where each and every
nucleic acid base of one strand is capable of forming hydrogen
bonds according to the canonical base pairing rules, with a
corresponding base in another, antiparallel strand. For example,
the sequences 5'-CTGACAATCG-3' and 5'-CGATTGTCAG-3' are totally
complementary (also referred to herein as "completely
complementary") to each other. As used herein "complete
complementarity" or "completely complementary" indicates that all
the contiguous residues of a nucleic acid sequence can form
Watson-Crick base pairing with the same number of contiguous
residues in a second nucleic acid sequence in an anti-parallel
fashion. A sequence that is completely complementary to another
sequence is also referred to as the complete complement of the
other.
[0065] The skilled artisan will appreciate that where there are no
bases that can adequately base pair with corresponding contiguous
residues in an antiparallel strand, the two strands would be
considered to have no complementarity. In certain embodiments
herein, at least portions of two antiparallel strands will have no
complementarity. In certain embodiments such portions may comprise
even a majority of the length of the two strands.
[0066] In addition to the foregoing, the skilled artisan will
appreciate that in strands of equal length that are completely
complementary, all sections of those strands are completely
complementary to each other. Strands which are not of equal length,
i.e. present in a nucleotide duplex having one or both ends not
being blunt, may be considered by those of skill in the art to be
completely complementary; however there will be one or more bases
in the overhanging end or ends ("overhangs") which do not have
corresponding bases in the opposing strand with which to base pair.
In the case of strands that are incompletely or partially
complementary, it is to be understood that there may be portions or
sections of the strands wherein there are several or even many
contiguous bases which are completely complementary to each other,
and other portions of the incompletely complementary strands which
have less than complete complementarity--i.e. those sections are
only partially complementary to each other.
[0067] The percentage of complementarity between a first nucleotide
sequence and a second nucleotide sequence can be evaluated by
sequence identity or similarity between the first nucleotide
sequence and the complement of the second nucleotide sequence. A
nucleotide sequence that is X % complementary to a second
nucleotide sequence is X % identical to the complement of the
second nucleotide sequence. The "complement of a nucleotide
sequence" is completely complementary to the nucleotide sequence,
whose sequence is readily deducible from the nucleotide sequence
using the rules of Watson-Crick base pairing.
[0068] "Conservation or conserved" indicates the extent to which a
specific sequence is found to be represented in a group of related
target sequences, regardless of the genetic context of the specific
sequence.
[0069] "Genetic context" refers to the flanking sequences that
surround a specific identified sequence and that are sufficiently
long to enable one of average skill in the art to determine its
position within a genome or RNA molecule relative to sequence
annotations or other markers in common use.
[0070] "Sequence identity or similarity", as known in the art, is
the relationship between two or more polypeptide sequences or two
or more polynucleotide sequences, as determined by comparing the
sequences. In the art, identity also means the degree of sequence
relatedness between polypeptide or polynucleotide sequences, as the
case can be, as determined by the match between strings of such
sequences. To determine the percent identity or similarity of two
amino acid sequences or of two nucleic acids, the sequences are
aligned for optimal comparison purposes (e.g., gaps can be
introduced in the sequence of a first amino acid or nucleic acid
sequence for optimal alignment with a second amino or nucleic acid
sequence). The amino acid residues or nucleotides at corresponding
amino acid positions or nucleotide positions are then compared.
When a position in the first sequence is occupied by the same or
similar amino acid residue or nucleotide as the corresponding
position in the second sequence, then the molecules are identical
or similar at that position. The percent identity or similarity
between the two sequences is a function of the number of identical
or similar positions shared by the sequences (i.e., %
identity=number of identical positions/total number of positions.
(e.g., overlapping positions).times.100). Two sequences that share
100% sequence identity are identical. In one embodiment, the two
sequences are the same length.
[0071] Both identity and similarity can be readily calculated.
Methods commonly employed to determine identity or similarity
between sequences include, but are not limited to those disclosed
in Carillo et al, (1988), SIAM J. Applied Math. 48, 1073. Preferred
methods to determine identity are designed to give the largest
match between the sequences tested. Methods to determine identity
and similarity are codified in computer programs.
[0072] A non-limiting example of a mathematical algorithm utilized
for the comparison of two sequences is the algorithm of Karlin et
al., (1990), Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as
in Karlin et al., (1993), Proc. Natl. Acad. Sci. USA 90:5873-5877.
Such an algorithm is incorporated into the NBLAST and XBLAST
programs of Altschul et al., (1990), J Mol. Biol. 215:403-410. To
obtain gapped alignments for comparison purposes, Gapped BLAST can
be utilized as described in Altschul et al., (1997), Nucleic Acids
Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform
an iterated search which detects distant relationships between
molecules. When utilizing BLAST, Gapped BLAST, and PSI-Blast
programs, the default parameters of the respective programs (e.g.,
XBLAST and NBLAST) can be used. Additionally, the FASTA method
(Atschul et al., (1990), J. Molec. Biol. 215, 403), can also be
used.
[0073] Another non-limiting example of a mathematical algorithm
useful for the comparison of sequences is the algorithm of Myers et
al, (1988), CABIOS 4:11-17. Such an algorithm is incorporated into
the ALIGN program (version 2.0).
[0074] In an embodiment, the percent identity between two sequences
is determined using the Needleman and Wunsch (J. Mol. Biol.
(48):444-453 (1970)) algorithm which has been incorporated into the
GAP program in the GCG software package. The Accelrys GCG GAP
program aligns two complete sequences to maximize the number of
matches and minimizes the number of gaps.
[0075] In another embodiment, the percent identity between two
sequences is determined using the local homology algorithm of Smith
and Waterman (J Mol Biol. 1981, 147(1):195-7), which has been
incorporated into the BestFit program in the Accelrys GCG software
package. The BestFit program makes an optimal alignment of the best
segment of similarity between two sequences. Optimal alignments are
found by inserting gaps to maximize the number of matches.
[0076] Nucleotide sequences that share a substantial degree of
complementarity will form stable interactions with each other, for
example, by matching base pairs. As used herein, the term "stable
interaction" with respect to two nucleotide sequences indicates
that the two nucleotide sequences have sufficient complementarity
and have the natural tendency to interact with each other to form a
double stranded molecule. Two nucleotide sequences can form stable
interaction with each other within a wide range of sequence
complementarity. In general, the higher the complementarity the
stronger or the more stable the interaction is. Different strengths
of interactions, may be required for different processes. For
example, the strength of interaction for the purpose of forming a
stable nucleotide sequence duplex in vitro may be different from
that for the purpose of forming a stable interaction between an
iRNA and a binding sequence in vivo. The strength of interaction
can be readily determined experimentally or predicted with
appropriate software by a person skilled in the art.
[0077] Hybridization can be used to test whether two
polynucleotides are substantially complementary to each other and
to measure how stable the interaction is. Polynucleotides that
share a sufficient degree of complementarity will hybridize to each
other under various hybridization conditions. In one embodiment,
polynucleotides that share a high degree of complementarity thus
form strong stable interactions and will hybridize to each other
under stringent hybridization conditions. "Stringent hybridization
conditions" has the meaning known in the art, as described in
Sambrook et al., Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,
(1989). An exemplary stringent hybridization condition comprises
hybridization in 6.times. sodium chloride/sodium citrate (SSC) at
about 45.degree. C., followed by one or more washes in
0.2.times.SSC and 0.1% SDS at 50-65.degree. C.
[0078] As used herein the term "mismatch" refers to a nucleotide of
either strand of two interacting strands having no corresponding
nucleotide on the corresponding strand or a nucleotide of either
strand of two interacting strands having a corresponding nucleotide
on the corresponding strand that is non-complementary.
[0079] As used herein, a "match" refers to a complementary pairing
of nucleotides.
[0080] As used herein, the term "expression system" refers to any
in vivo or in vitro system that can be used to evaluate the
expression of a target RNA molecule and or the RNAi activity of a
multitargeting RNA molecule of the invention. In particular
embodiments, the "expression system" comprises one or more target
RNA molecules, a multitargeting interfering RNA molecule targeting
the target RNA molecules, and a cell or any type of in vitro
expression system known to a person skilled in the art that allows
expression of the target RNA molecules and RNAi.
[0081] As used herein, the term "RNA" includes any molecule
comprising at least one ribonucleotide residue, including those
possessing one or more natural nucleotides of the following bases:
adenine, cytosine, guanine, and uracil; abbreviated A, C, G, and U,
respectively, modified ribonucleotides, universal base, acyclic
nucleotide, abasic nucleotide and non-ribonucleotides.
"Ribonucleotide" means a nucleotide with a hydroxyl group at the 2'
position of a p-D-ribo-furanose moiety.
[0082] As used herein, the term "non-target transcriptome" or
"non-targeted transcriptome" indicates the transcriptome aside from
the targeted RNA molecules. For example, when a multitargeting
interfering RNA is designed to target a viral RNA, the non-targeted
transcriptome is that of the host. When a multitargeting
interfering RNA is designed to target a given RNA in a biological
system, the non-targeted transcriptome is the transcriptome of the
biological system aside from the given targeted RNA.
[0083] Modified ribonucleotides include, for example 2'deoxy,
2'deoxy-2'-fluoro, 2'O-methyl, 2'O-methoxyethyl, 4'thio or locked
nucleic acid (LNA) ribonucleotides. Also contemplated herein is the
use of various types of ribonucleotide analogues, and RNA with
internucleotide linkage (backbone) modifications. Modified
internucleotide linkages include for example,
phosphorothioate-modified, and even inverted linkages (i.e. 3'-3'
or 5'-5'). Preferred ribonucleotide analogues include
sugar-modified, and nucleobase-modified ribonucleotides, as well as
combinations thereof. In preferred sugar-modified ribonucleotides
the 2'-OH-group is replaced by a substituent selected from H, OR,
R, halo, SH, SR, NH.sub.2, NHR, NR.sub.2 or ON, wherein R is C1-C6
alkyl, alkenyl or alkynyl and halo is F, Cl, Br, or I. In preferred
backbone-modified ribonucleotides, the phosphoester group
connecting to adjacent ribonucleotides is replaced by a modified
group, e.g. a phosphorothioate group. Any or all of the above
modifications may be combined. In addition, the 5' termini can be
OH, phosphate, diphosphate or triphosphate. Nucleobase-modified
ribonucleotides, i.e. ribonucleotides wherein the
naturally-occurring nucleobase is replaced with a non-naturally
occurring nucleobase instead, for example, uridines or cytidines
modified at the S-position (e.g. 5-(2-amino)propyl uridine, and
5-bromo uridine); adenosines and guanosines modified at the
8-position (e.g. 8-bromo guanosine); deaza nucleotides (e.g.
7-deaza-adenosine); O- and N-alkylated nucleotides (e.g. N6-methyl
adenosine) are also contemplated for use herein.
[0084] The term "universal base" as used herein refers to
nucleotide base analogs that form base pairs with each of the
natural DNA/RNA bases with little discrimination between them.
Non-limiting examples of universal bases include C-phenyl,
C-naphthyl and other aromatic derivatives, inosine, azole
carboxamides, and nitroazole derivatives such as 3-nitropyrrole,
4-nitroindole, 5-nitroindole, and 6-nitroindole as known in the art
(see for example Loakes, 2001, Nucleic Acids Research, 29,
2437-2447).
[0085] The term "acyclic nucleotide" as used herein refers to any
nucleotide having an acyclic ribose sugar, for example where any of
the ribose carbons (C1, C2, C3, C4, or C5), are independently or in
combination absent from the nucleotide.
[0086] As used herein with respect to the listing of RNA sequences,
the bases thymidine ("T") and uridine ("U") are frequently
interchangeable depending on the source of the sequence information
(DNA or RNA). Therefore, in disclosure of target sequences, seed
sequences, candidate seeds, target RNA binding sites, and the like,
the base "T" is fully interchangeable with the base "U". However,
with respect to specific disclosures of the interfering RNA
molecules of the invention, it is to be understood that for such
sequences the use of the base "U" cannot be generally substituted
with "T" in a functional manner. It is however known in the art
that certain occurrences of the base "U" in RNA molecules can be
substituted with "T" without substantially deleterious effect on
functionality. For example, the substitution of T for U in
overhangs, such as UU overhangs at the 3' end is known to be
silent, or at a minimum, acceptable, and thus is permissible in the
interfering RNA sequences provided herein. Thus, it is contemplated
that the skilled artisan will appreciate how to vary even the
specific interfering RNA sequences disclosed herein to arrive at
other structurally-related and functionally-equivalent structures
that are within the scope of the instant invention and the appended
claims.
[0087] A "target RNA molecule" or a "pre-selected target RNA
molecule" as used herein refers to any RNA molecule whose
expression or activity is desired to be modulated, for example
decreased, by an interfering RNA molecule of the invention in an
expression system. A "target RNA molecule" can be a messenger RNA
molecule (mRNA) that encodes a polypeptide of interest. A messenger
RNA molecule typically includes a coding region and non-coding
regions preceding ("5'UTR") and following ("3'UTR") the coding
region. A "target RNA molecule" can also be a non-coding RNA
(ncRNA), such as small temporal RNA (stRNA), micro RNA (miRNA),
small nuclear RNA (snRNA), short interfering RNA (siRNA), small
nucleolar RNA (snoRNA), ribosomal RNA (rRNA), transfer RNA (tRNA)
and precursor RNAs thereof. Such non-coding RNAs can also serve as
target RNA molecules because ncRNA is involved in functional or
regulatory cellular processes. Aberrant ncRNA activity leading to
disease can therefore be modulated by multitargeting interfering
RNA molecules of the invention. The target RNA can further be the
genome of a virus, for example a RNA virus, or a replicative
intermediate of any virus at any stage, as well as any combination
of these.
[0088] The "target RNA molecule" can be a RNA molecule that is
endogenous to a biological system, or a RNA molecule that is
exogenous to the biological system, such as a RNA molecule of a
pathogen, for example a virus, which is present in a cell after
infection thereof. A cell containing the target RNA can be derived
from or contained in any organism, for example a plant, animal,
protozoan, virus, bacterium, or fungus. Non-limiting examples of
plants include monocots, dicots, or gymnosperms. Non-limiting
examples of animals include vertebrates or invertebrates.
Non-limiting examples of fungi include molds or yeasts.
[0089] A "target RNA molecule" as used herein may include any
variants or polymorphism of a desired RNA molecule. Most genes are
polymorphic in that a low but nevertheless significant rate of
sequence variability occurs in a gene among individuals of the same
species. Thus, a RNA molecule may correlate with multiple sequence
entries, each of which represents a variant or a polymorphism of
the RNA molecule. In designing any gene suppression tool there is
the risk that the selected binding sequence(s) used in the
computer-based design may contain relatively infrequent alleles. As
a result, the active sequence designed might be expected to provide
the required benefit in only a small proportion of individuals. The
frequency, nature and position of most variants (often referred to
as single nucleotide polymorphisms (SNPs)) are easily accessible to
those trained in the art. In this respect, sequences with a target
molecule that are known to be highly polymorphic can be avoided in
the selection of binding sequences during the bioinformatic screen.
Alternatively, a limitless number of sequences available for any
particular target may be used in the design stages of an
interfering RNA of the invention to make sure that the targeted
binding sequence is present in the majority of allelic variants,
with the exception of the situation in which targeting of the
allelic variant is desired (that is, when the allelic variant
itself is implicated in the disease of interest).
[0090] A "target RNA molecule" comprises at least one targeted
binding sequence that is sufficiently complementary to the guide
sequence of an interfering RNA molecule of the invention to allow
stable interaction of the binding sequence with the guide sequence.
The targeted binding sequence can be refined to include any part of
the transcript sequence (eg 5'UTR, ORF, 3'UTR) based on the desired
effect. For example, translational repression is a frequent
mechanism operating in the 3'UTR (i.e. as for microRNA). Thus, the
targeted binding sequence can include sequences in the 3' UTR for
effective translational repression.
[0091] The "targeted binding sequence", "binding sequence", or
"target sequence" shall all mean a portion of a target RNA molecule
sequence comprising a seed sequence and the sequence flanking
either one or both ends of the seed, said binding sequence
predicted to a form stable interaction with one strand of a
multitargeting interfering RNA of the invention based on the
complementarity between the said strand and the binding
sequence.
[0092] As used herein the term "seed" or "seed sequence" or "seed
region sequence" refers to a sequence of at least about 6
contiguous nucleotides present in a target RNA that is completely
complementary to a portion of one strand of an interfering RNA.
Although 6 or more contiguous bases are preferred, the expression
"about 6" refers to the fact that windows of at least 5 or more
contiguous bases or more can provide useful candidates in some
cases and can ultimately lead to the design of useful interfering
RNAs. Thus, all such seed sequences are contemplated within the
scope of the instant invention.
[0093] As used herein, the term "interfering RNA" or "iRNA" is used
to indicate single or double stranded RNA molecules that modulate
the presence, processing, transcription, translation, or half-life
of a target RNA molecule, for example by mediating RNA interference
("RNAi"), in a sequence specific manner. As used herein, the term
"RNA interference" or "RNAi" is meant to be equivalent to other
terms used to describe sequence specific RNA interference, such as
post-transcriptional gene silencing, translational inhibition, or
epigenetics. This includes, for example, RISC-mediated degradation
or translational repression, as well as transcriptional silencing,
altered RNA editing, competition for binding to regulatory
proteins, and alterations of mRNA splicing. It also encompasses
degradation and/or inactivation of the target RNA by other
processes known in the art, including but not limited to
nonsense-mediated decay, and translocation to P bodies. Thus, the
interfering RNAs provided herein (e.g. CODEMIRs and VIROMIRs) may
exert their functional effect via any of the foregoing mechanisms
alone, or in combination with one or more other means of RNA
modulation known in the art. The interfering RNAs provided herein
can be used to manipulate or alter the genotype or phenotype of an
organism or cell, by intervening in cellular processes such as
genetic imprinting, transcription, translation, or nucleic acid
processing (e.g., transamination, methylation, etc.).
[0094] The term "interfering RNA" is meant to be equivalent to
other terms used to describe nucleic acid molecules that are
capable of mediating sequence specific RNAi, for example short
interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA
(miRNA), short hairpin RNA (shRNA), short interfering
oligonucleotide, short interfering nucleic acid, short interfering
modified oligonucleotide, chemically-modified siRNA,
post-transcriptional gene silencing RNA (ptgsRNA), and others.
[0095] The "interfering RNA" can be assembled from two separate
oligonucleotides. The "interfering RNA" can also be assembled from
a single oligonucleotide, comprising self-complementary regions
linked by means of a nucleic acid based or non-nucleic acid-based
linker(s). The "interfering RNA" can be a polynucleotide with a
duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary
structure, having self-complementary regions. The "interfering RNA"
can also be a single-stranded polynucleotide having one or more
loop structures and a stem comprising self-complementary regions
(e.g. short hairpin RNA, shRNA), wherein the polynucleotide can be
processed either in vivo or in vitro to generate one or more double
stranded interfering RNA molecules capable of mediating RNA
inactivation. The cleavage of the self-paired region or regions of
the single strand RNA to generate double-stranded RNA can occur in
vitro or in vivo, both of which are contemplated for use
herein.
[0096] As used herein, the "interfering RNA" need not be limited to
those molecules containing only RNA, but further encompasses those
possessing one or more modified ribonucleotides and
non-nucleotides, such as those described supra.
[0097] The term "interfering RNA" includes double-stranded RNA,
single-stranded RNA, isolated RNA such as partially purified RNA,
essentially pure RNA, synthetic RNA, recombinantly produced RNA, as
well as altered RNA that differs from naturally occurring RNA by
the addition, deletion, substitution and/or alteration of one or
more nucleotides. Such alterations can include addition of
non-nucleotide material, such as to the end(s) of the
multitargeting interfering RNA or internally, for example at one or
more nucleotides of the RNA. Nucleotides in the RNA molecules of
the instant invention can also comprise non-standard nucleotides,
such as non-naturally occurring nucleotides or chemically
synthesized nucleotides or deoxynucleotides. These altered RNAs can
be referred to as analogs or analogs of naturally occurring
RNA.
[0098] The interfering RNA of the invention, also termed
"multitargeting interfering RNA" is an interfering double-stranded
RNA, each strand of which can form stable interactions with binding
sites present in distinct genetic contexts on one or more target
RNA molecules. Examples of the multitargeting interfering
RNA.include CODEMIRs, COmputationally-DEsigned, Multi-targeting
Interfering RNAs, and VIROMIRs, where the latter multitargeting
interfering RNA molecules are preferentially targeted to viral
targets.
[0099] "Sequence" means the linear order in which monomers occur in
a polymer, for example, the order of amino acids in a polypeptide
or the order of nucleotides in a polynucleotide.
[0100] A "subject" as used herein, refers to an organism to which
the nucleic acid molecules of the invention can be administered. A
subject can be an animal or a plant, preferably a mammal, most
preferably a human, who has been the object of treatment,
observation or experiment, or any cell thereof.
[0101] A "vector" refers to a nucleic acid molecule capable of
transporting another nucleic acid to which it has been linked. One
type of vector is a "plasmid", which refers to a circular double
stranded DNA loop into which additional DNA segments can be
inserted. Another type of vector is a viral vector, wherein
additional DNA segments can be inserted. Certain vectors are
capable of autonomous replication in a host cell into which they
are introduced (e.g., bacterial vectors having a bacterial origin
of replication and episomal mammalian vectors). Other vectors
(e.g., non-episomal mammalian vectors) are integrated into the
genome of a host cell upon introduction into the host cell, and
thereby are replicated along with the host genome. Moreover,
certain vectors, expression vectors, are capable of directing the
expression of genes to which they are operably linked.
[0102] As used herein, "modulate (or modulation of) the expression
of an RNA molecule" means any RNA interference mediated regulation
of the level and/or biological activity of the RNA molecule. It
includes any RNAi-related post-transcriptional gene silencing, such
as by cleaving, destabilizing the target RNA molecule or preventing
their translation. In one embodiment, the term "modulate" can mean
"inhibit," but the use of the word "modulate" is not limited to
this definition. The modulation of the target RNA molecule is
determined in a suitable expression system, for example in vivo, in
one or more suitable cells, or in an acellular or in vitro
expression system such as are known in the art. Routine methods for
measuring parameters of the transcription, translation, or other
aspects of expression relating to RNA molecules are known in the
art, and any such measurements are suitable for use herein.
[0103] By "inhibit", "down-regulate", "reduce", or "decrease" or
"decreasing" as with respect to a target RNA or its expression it
is meant that the expression of the gene or level and/or biological
activity of target RNA molecules is reduced below that observed in
the absence of the nucleic acid molecules (e.g., multitargeting
interfering RNA) of the invention. In one embodiment, inhibition,
down-regulation or reduction with a multitargeting interfering RNA
molecule is greater than that observed in the presence of an
inactive or attenuated molecule. In another embodiment, inhibition,
down-regulation, or reduction with a multitargeting interfering RNA
molecule is greater than that observed in the presence of, for
example, multitargeting interfering RNA molecule with scrambled
sequence or with mismatches. In another embodiment, inhibition,
down-regulation, or reduction of gene expression with a nucleic
acid molecule of the instant invention is greater in the presence
of the nucleic acid molecule than in its absence.
[0104] "Inhibit", "down-regulate", "reduce", or "decrease" as with
respect to a target RNA or its expression encompasses, for example,
reduction of the amount or rate of transcription or translation of
a target RNA, reduction of the amount or rate of activity of the
target RNA, and/or a combination of the foregoing in a selected
expression system. The skilled artisan will appreciate that a
decrease in the total amount of transcription, the rate of
transcription, the total amount of translation, or the rate of
translation, or even the activity of an encoded gene product are
indicative of such a decrease. The "activity" of an RNA refers to
any detectable effect the RNA may have in a cell or expression
system, including for example, any effect on transcription, such as
enhancing or suppressing transcription of itself or another RNA
molecule. The measurement of a "decrease" in expression or the
determination of the activity of a given R NA can be performed in
vitro or in vivo, in any system known or developed for such
purposes, or adaptable thereto. Preferably the measurement of a
"decrease" in expression by a particular interfering RNA is made
relative to a control, for example, in which no interfering RNA is
used. In some comparative embodiments such measurement is made
relative to a control in which some other interfering RNA or
combination of interfering RNAs is used. Most preferably a change,
such as the decrease is statistically significant based on a
generally accepted test of statistical significance. However,
because of the large number of possible measures and the need for
the ability to rapidly screen candidate interfering RNAs, it is
contemplated herein that a given RNA need only show an arithmetic
decrease in one such in vitro or in vivo assay to be considered to
show a "decrease in expression" as used herein.
[0105] More particularly, the biological modulating activity of the
multitargeting interfering RNA is not limited to, or necessarily
reliant on, degradation or translational repression by conventional
RISC protein complexes involved in siRNA and microRNA
gene-silencing, respectively. Indeed, short double-stranded and
single-stranded RNA have been shown to have other possible
sequence-specific roles via alternative mechanisms. For example,
short double-stranded RNA (dsRNA) species may act as modulatory
effectors of differentiation/cell activity, possibly through
binding to regulatory proteins (Kuwabara, T., et al., (2004), Cell,
116: 779-93). Alternatively, dsRNA may lead to the degradation of
mRNA through the involvement of AU-rich element (ARE)-binding
proteins (Jing, Q., et al., (2005), Cell, 120: 623-34). Further,
dsRNA may also induce epigenetic transcriptional silencing (Morris,
K. V., et al., (2004) Science, 305: 1289-89). Processing of mRNA
can also be altered through A to I editing and modified
splicing.
[0106] As used herein, "palindrome" or "palindromic sequence" means
a nucleic acid sequence that is completely complementary to a
second nucleotide sequence that is identical to the nucleic acid
sequence, e.g., UGGCCA. The term also includes a nucleic acid
molecule comprising of two nucleotide sequences that are
palindromic sequences.
[0107] "Phenotypic change" as used herein refers to any detectable
change to a cell or an organism that occurs in response to contact
or treatment with a nucleic acid molecule of the invention. Such
detectable changes include, but are not limited to, changes in
shape, size, proliferation, motility, protein expression or RNA
expression or other physical or chemical changes as can be assayed
by methods known in the art. The detectable change can also include
expression of reporter genes/molecules such as Green Fluorescent
Protein (GFP) or various tags that are used to identify an
expressed protein or any other cellular component that can be
assayed.
[0108] The term "therapeutically effective amount" as used herein,
means that amount of active compound or pharmaceutical agent that
elicits the biological or medicinal response in a tissue system,
animal, human, or plant that is being sought by a researcher,
veterinarian, medical doctor or other clinician, which includes
ameliorating or alleviation of the symptoms of the disease or
disorder being treated. Methods are known in the art for
determining therapeutically effective doses for the instant
pharmaceutical composition.
[0109] The term "prophylactically effective amount" refers to that
amount of active compound or pharmaceutical agent that inhibits in
a subject the onset of a disorder as being sought by a researcher,
veterinarian, medical doctor or other clinician.
[0110] In general, the interfering RNAs known to one of ordinary
skill in the art are double-stranded polynucleotide molecules
comprising two self-complementary strands which are sense and
antisense to the target. The iRNA duplex is usually designed such
that the antisense (guide) strand is preferentially loaded into the
RISC and guides the RISC-mediated degradation of the target
nucleotide sequence following complementary base-pairing. The sense
(passenger) strand may be degraded in the process of loading into
the RISC complex or soon after by endonucleases to which single
stranded RNA is highly sensitive. The relative thermodynamic
characteristics of the 5' termini of the two strands of an
interfering RNA determine whether a strand serves the function of a
passenger or a guide strand during RNAi.
[0111] The present invention provides a multitargeting interfering
RNA molecule comprising two strands, each of which is designed
against a specific target sequence. The iRNA duplex is designed in
such a manner that each strand can be loaded into RISC complexes
and thus both strands function as "guide" strands. Preferably both
strands are loaded to an approximately equal extent into RISC
complexes. One strand is at least partially complementary to a
first portion of a target RNA binding sequence, which is also
referred to as the seed. The other strand comprises a sequence
which is at least partially if not completely identical to the
seed, this sequence being at least partially complementary to the
first portion of a second target RNA binding sequence. The said
first and second target binding sequences are present in distinct
genetic contexts in at least one pre-selected target RNA molecule.
That is, multiple target RNA binding sites may be present on the
same target RNA molecule, on separate RNA molecules, or both.
[0112] In one general aspect, the present invention provides a
multitargeting interfering RNA molecule comprising Formula (I):
5'-p-XSY-3' 3'-X'S'Y'-p-5'
[0113] In Formula (I), p consists of a terminal phosphate group
that can be present or absent from the 5'-end of either strand. Any
terminal phosphate group known to a person skilled in the art can
be used. Such phosphate group includes, but is not limited to,
monophosphate, diphosphate, triphosphate, cyclic phosphate or to a
chemical derivative of phosphate such as a phosphate ester
linkage.
[0114] In Formula (I), S consists of a first nucleotide sequence of
a length of about 5 to about 20 nucleotides that is completely
complementary to a first portion of a first binding sequence, and
S' consists of a second nucleotide sequence of a length of about 5
to about 20 nucleotides that is completely complementary to a first
portion of a second binding sequence, wherein said first and second
binding sequences are present in distinct genetic contexts in at
least one pre-selected target RNA molecule, and wherein S and S'
are at least substantially complementary to each other but are not
palindromic.
[0115] In particular embodiments, S and S' each has a length of,
for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20 nucleotides that are at least partially, preferably
completely, complementary to the first portion of the at least two
binding sequences. In one embodiment, S is completely complementary
to a sequence present in one or more pre-selected target RNA
molecules. In another embodiment, S' is completely complementary to
a sequence present in one or more pre-selected target RNA
molecules. In particular embodiments, S and S' are completely
complementary to each other.
[0116] In certain embodiments, S is partially complementary to a
first portion of a binding sequence present in one or more
pre-selected target RNA molecules, such as 6 of 7, 7 of 8, 8 of 9,
9 of 10, 10 of 11, 11 of 12, 12 of 13, 13 of 14, 14 of 15, or 15 of
16 consecutive nucleotides of S are completely complementary to the
first portion of at least one target RNA binding sequence. In other
embodiments, S and the first portion of the distinct binding
sequences have lesser overall complementarity such as 10 of 12, 11
of 13, 12 of 14, 13 of 15, or 14 of 16 nucleotides of complete
complementarity. Similarly, in certain embodiments, S' is partially
complementary to a first portion of a second binding site.
[0117] The remaining sequence of the two strands of the
multitargeting interfering RNA (X,X', Y and Y') in Formula (I) is
independently absent or consists of a nucleotide sequence. In
particular embodiments, they are developed so as to generate
further binding to the target RNA sites. In one embodiment, the
sequences of X and Y' are at least partially complementary to the
second portions of the first and second target RNA binding
sequences, respectively. In one embodiment, the sequences X' and Y
are completely complementary to X and Y', respectively, such that
XSY and Y'S'X' are completely complementary. In an additional
embodiment, X' and Y are incompletely complementary with X and Y',
respectively such that XSY and Y'S'X' are incompletely
complementary. This may be required, for example, in situations in
which the loading bias of the interfering RNA duplex needs to be
altered through the use of mismatches in the extremity with the
higher hybridization energy.
[0118] In a further embodiment, the sequences X and Y' are designed
so as to maximize binding of XS and Y'S' to the first and second
portions of a plurality of target RNA binding sites. In this
situation, the plurality of target sequences (e.g. viral isolates)
can be examined in order to generate a number of identity consensus
sequences corresponding to the second portion of the plurality of
target RNA sequences. These identity consensus sequences can be
generated by hand by examining the alignments of the target RNA
sequences. Alternatively, all possible base sequences or a subset
of putative XS and Y'S' sequences can be generated by computer
algorithm. Each putative XS and Y'S' sequence is then hybridized in
silico using RNAhybrid or a similar program known to one skilled in
the art. Those putative sequences that are predicted to best bind
the corresponding first and second portions of the target RNA
binding sites are then prioritized for the next design phase, which
includes filtering out putative sequences that have unfavorable
characteristics such as more than 4 contiguous C or G bases.
[0119] In a preferred embodiment, the sequences of Y and X' are
then designed such that they are at least partially complementary
to Y' and X, respectively. Overhangs, if required may simply be the
addition to X' and Y of UU, dTdT or any other base or modified
base. In one embodiment, the bases of the overhangs are selected so
as to further increase the predicted binding of XSY and Y'S'X' to
their respective RNA targets. Overhangs may be 1, 2, 3, 4 or 5
bases as required.
[0120] In an interfering RNA of the invention, a preferred
embodiment is one in which the two strands of the duplex
independently have either partial or complete complementarity to
their corresponding at least one target sequence and the two
strands are completely complementary to one another, excepting the
overhangs when present. Another embodiment of the invention is one
in which each of the two strands of the duplex independently have
either partial or complete complementary to their corresponding at
least one target sequence and the two strands are incompletely
complementary to one another. Both strands can be modified and
refined to enhance some aspect of the function of the interfering
RNA molecule of the invention. For example, various pharmacophores,
dyes, markers, ligands, conjugates, antibodies, antigens, polymers,
peptides and other molecules can be conveniently linked to the
molecules of the invention. The interfering RNA can further
comprise one or more 5' terminal phosphate group, such as a
5'-phosphate or 5',3'-diphosphate. These may be of use to improve
cell uptake, stability, tissue targeting or any combination
thereof.
[0121] In another embodiment, X consists of a nucleotide sequence
that is at least partially complementary to a second portion of the
first binding sequence, said second portion is adjacent to and
connected with the 3'-end of said first portion of the first
binding sequence, and wherein X' is substantially complementary to
X. In a particular embodiment, X and X' are completely
complementary to each other. In another particular embodiment, X is
completely complementary to the second portion of the first binding
sequence.
[0122] In yet another embodiment, Y' consists of a nucleotide
sequence that is at least partially complementary to a second
portion of the second binding sequence, said second portion is
adjacent to and connected with the 3'-end of said first portion of
the second binding sequence, and wherein Y is substantially
complementary to Y'. In a particular embodiment, Y and Y' are
completely complementary to each other. In another particular
embodiment, Y' is completely complementary to the second portion of
the second binding sequence.
[0123] In Formula (I), XSY is at least partially complementary to
the first binding sequence to allow stable interaction of XSY with
the first binding sequence, and Y'S'X' is at least partially
complementary to the second binding sequence to allow stable
interaction with the second binding sequence, and XSY and Y'S'X'
are at least partially complementary to each other to allow
formation of a stable iRNA duplex. In a particular embodiment, XSY
is completely complementary to the first binding sequence. In
another embodiment, Y'S'X' is completely complementary to the
second binding sequence. In yet another embodiment, XSY and Y'S'X'
are completely complementary to each other.
[0124] In an embodiment of the present invention, each strand of a
multitargeting interfering RNA molecule of the invention is
independently about 17 to about 25 nucleotides in length, in
specific embodiments about 17, 18, 19, 20, 21, 22, 23, 24, and 25
nucleotides in length. Using shorter length interfering RNA
molecules without the need for the generation of multiple active
sequences through processing of RNA by enzymes such as Dicer and
RNaseIII, provides advantages, for example, in reduction of cost,
manufacturing, and chance of off-target effects.
[0125] The interaction between the two strands can be adjusted to
improve loading of both strands into the cellular RISC complex
(Khvorova et al. (2003) Cell, 115: 209-16; Schwarz et al. (2003)
Cell, 115: 199-208), or to otherwise improve the functional aspects
of the interfering RNA. The skilled artisan will appreciate that
there are routine methods for altering the strength and other
properties of the base paired strands through the addition,
deletion, or substitution of one or more bases in either strand of
the synthetic duplex. In particular as one example, these
strategies can be applied to the design of the extremities of the
duplex to ensure that the predicted thermodynamics of the duplex
are conducive to the loading of the desired strand. These
strategies are well known to persons skilled in the art.
[0126] It is also contemplated herein that a single-stranded RNA
molecule comprises, for example, a hairpin loop or similar
secondary structure that allows the molecule to self-pair to form
at least a region of double-stranded nucleic acid of Formula
(I).
[0127] The skilled artisan will appreciate that the double-stranded
RNA molecules provide certain advantages for use in therapeutic
applications. Although blunt-ended molecules are disclosed herein
for certain embodiments, in various other embodiments, overhangs,
for example of 1-5 nucleotides, are present at either or both
termini. In some embodiments, the overhangs are 2 or 3 bases in
length. Presently preferred overhangs include 3'-terminus UU
overhangs (3'-UU) in certain embodiments. Other overhangs
exemplified for use herein include, but are not limited to, 3'-AA,
3'-CA, 3'-AU, 3'-UC, 3'-CU, 3'-UG, 3'-CC, 3'-UA, 3'-U, and 3'-A.
Still other either 5'-, or more preferably 3'-, overhangs of
various lengths and compositions are contemplated for use herein on
the RNA molecules provided.
[0128] In certain embodiments, the multitargeting interfering RNA
molecule of the invention comprises one or more terminal overhangs,
for example, an overhang consisting 1 to 5 nucleotides. In other
embodiments, the multitargeting interfering RNA molecule of the
invention comprises at least one modified ribonucleotide, such as
one 2'-O-methyl ribosyl substitution.
[0129] In certain embodiments at least one target RNA molecule is
an mRNA. More specifically, in some embodiments at least one target
encodes a receptor, cytokine, transcription factor, regulatory
protein, signaling protein, cytoskeletal protein, transporter,
enzyme, hormone, or antigen. As such, the potential range of
protein targets in the cell is not limited, however the skilled
artisan will appreciate that certain targets are more likely to be
of value in a particular disease state or process. In addition, the
skilled artisan will appreciate that target RNA molecules, whether
coding or regulatory, originating from a pathogen (e.g. a virus)
are useful with the multitargeting interfering RNAs and methods
provided herein.
[0130] In one embodiment, at least one of the binding sequences is
in the 3' UTR of an mRNA.
[0131] The inclusion of one target or more targets does not
preclude the use of, or intention for, a particular interfering RNA
to target another selected target. Such targeting of any additional
RNA target molecules may result in less, equal, or greater effect
in an expression system. Notwithstanding the foregoing, the
multitargeting interfering RNAs of the instant invention are
preferably screened for off-target effects, especially those that
are likely. For example, reviewing the potential binding to the
entire transcriptome, or as much of it as is known at the time
provides a useful approach to such screening. For example, where a
genome has been completely sequenced, the skilled artisan will
appreciate that the entire transcriptome can be conveniently
screened for likely off-target effects. In cases for which local
delivery of multitargeting interfering RNA is anticipated,
specialized tissue-specific transcriptomes (eg retina for ocular
applications) may be more relevant because non-target transcripts
that are identified through bioinformatic approaches from the
complete transcriptome may actually not be present in the tissue
into which the multitargeting interfering RNA is applied.
[0132] In one embodiment, the two strands of a multitargeting
interfering RNA of the invention form stable interaction with at
least two distinct targeted binding sequences present in distinct
genetic contexts on a single target RNA molecule, and thus
modulates the expression or activity of the RNA molecule. Targeting
multiple binding sites on a single target RNA molecule with a
single iRNA provides more effective RNAi of the target RNA
molecule. This approach is particularly useful for the modulation
of virus gene expression where the mutation rate is high.
[0133] In another embodiment, the two strands of a multitargeting
interfering RNA of the invention form stable interaction with at
least two binding sequences present in distinct genetic contexts on
multiple pre-selected target RNA molecules, and thus modulates the
expression or activity of multiple pre-selected target RNA
molecules. Targeting multiple target RNA molecules with a single
iRNA represents an alternative to the prototypical one drug one
target approach. In considering the complexity of biological
systems, designing a drug selective for multiple targets will lead
to new and more effective medications for a variety of diseases and
disorders.
[0134] In specific embodiments, RNA molecules that are involved in
a disease or disorder of a biological system are pre-selected and
targeted by a multitargeting interfering RNA molecule of the
invention. The biological system can be, for example, a plant, or
an animal such as a rat, a mouse, a dog, a pig, a monkey, and a
human. The pre-selected target RNA molecules can, for example,
encode a protein of a class selected from the group consisting of
receptors, cytokines, transcription factors, regulatory proteins,
signaling proteins, cytoskeletal proteins, transporters, enzymes,
hormones, and antigens. The pre-selected target RNA molecules can,
for example, encode a protein selected from the group consisting of
ICAM-1, VEGF-A, MCP-1, IL-8, VEGF-B, IGF-1, Gluc6p, Inppl1, bFGF,
PlGF, VEGF-C, VEGF-D, .beta.-catenin, .kappa.-ras-B, .kappa.-ras-A,
EGFR, and TNF alpha. Therefore, the multitargeting interfering RNA
molecule of the invention can, for example, modulate expression of
any combination of ICAM-1, VEGF-B, VEGF-C, VEGF-D, IL-8, bFGF,
PlGF, MCP-1 and IGF-1, any combination of ICAM-1, VEGF-A and IGF-1,
any combination of .beta.-catenin, .kappa.-ras, and EGFR, both
ICAM-1 and VEGF-A, or both Gluc6p and Inppl1, in a biological
system, such as an animal.
[0135] The pre-selected target RNA molecule can also be a viral
RNA, including a viral RNA encoding a protein essential for the
virus. Such essential proteins can, for example, be involved in the
replication, transcription, translation, or packaging activity of
the virus. Exemplary essential proteins for a HIV virus are GAG,
POL, VIF, VPR, TAT, NEF, REV, VPU and ENV, all of which can be a
pre-selected target molecule of the invention. The multitargeting
interfering RNA of the invention can be used to modulate viral RNA
from, including but not limited to, a human immunodeficiency virus
(HIV), a hepatitis C virus (HCV), an influenza virus, a rhinovirus,
and a severe acute respiratory syndrome (SARS) virus or a
combination thereof.
[0136] In some embodiments, the multitargeting interfering RNA of
the invention are designed to target one or more target RNA
molecules in a first biological system and one or more target
molecules in a second biological system that is infectious to the
first biological system. In particular embodiments, the
multitargeting interfering RNA of the invention are designed to
target one or more host RNA molecules and one or more RNA molecules
of a virus or a pathogen for the host. In particular embodiments of
the invention, the viral RNA is HCV or HIV and the host target RNA
includes, but is not limited to, TNFalpha, LEDGF(p75), BAF, CCR5,
CXCR4, furin, NFkB or STAT1.
[0137] In particular embodiments of the invention, specific
multitargeting interfering RNA molecules are provided in the
Examples that are functional against specific targets. These
CODEMIRs and/or VIROMIRs are useful for decreasing expression of
RNAs, for example, their intended target RNA molecules and data
supporting the activity are also provided herein in the working
examples. Such molecules, the skilled artisan will appreciate, can
target multiple sites on a single RNA or multiple sites on two or
more RNAs and are useful to decrease the expression of such one or
preferably two or more such targeted RNAs in an expression
system.
[0138] In some embodiments, a given multitargeting interfering RNA
will be more effective at modulating expression of one of several
target RNAs than another. In other cases, the multitargeting
interfering RNA will similarly affect all targets in one or more
expression systems. Various factors can be responsible for causing
variations in silencing or RNAi efficiency: (i) asymmetry of
assembly of the RISC causing one strand to enter more efficiently
into the RISC than the other strand; (ii) inaccessibility of the
targeted segment on the target RNA molecule; (iii) a high degree of
off-target activity by the interfering RNA; (iv) sequence-dependent
variations for natural processing of RNA, and (v) the balance of
the structural and kinetic effects described in (i) to (iv). See
Hossbach et al. (2006), RNA Biology 3: 82-89. In designing a
multitargeting interfering RNA molecule of the invention, special
attention can be given to each of the listed factors to increase or
decrease the RNAi efficiency on a given target RNA molecule.
[0139] Another general aspect of the invention is a method for
designing a multitargeting interfering RNA. The method of the
invention includes various means leading to a multitargeting
interfering RNA that effectively target distinct binding sequences
present in distinct genetic contexts in one or more pre-selected
target RNA molecules. In one embodiment, a multitargeting
interfering RNA can be designed by visual or computational
inspection of the sequences of the target molecules, for example,
by comparing target sequences and their complements and identifying
sequences of length n which occur in both the target sequence and
the complement of the target sequence sets. In another embodiment,
a multitargeting interfering RNA can be designed by visual or
computational inspection of the sequences of the target molecules
to find occurrences of the sequence of length n and of its complete
complement within the set of target sequences. Alternatively, all
possible sequences of a pre-selected length n can be generated by
virtue of each permutation possible for each nucleotide position to
a given length (4.sup.n) and then examining for their occurrence in
the pre-selected nucleotide sequences and their complements.
Alternatively, all possible sequences of a pre-selected length n
and their complete complements can be generated by virtue of each
permutation possible for each nucleotide position to a given length
(4.sup.n) and then examining for their occurrence in the
pre-selected nucleotide sequences.
[0140] In one embodiment, when there is a pluarilty of target
sequences, a multitargeting interfering RNA can be designed by
visual or computational inspection of the sequences of the target
molecules, for example, by aligning sequences and visually or
computationally finding consensus target sequences for the
design.
[0141] For both strands of a given multitargeting interfering RNA
molecule of the invention to be active, the rational design process
requires that each strand be capable of modulating expression of
its intended target (i.e. each strand is "active" against its
target RNA, e.g. by having at least partial complementarity
thereto) while simultaneously requiring that each of the strands is
at least sufficiently complementary to the other that a duplex can
form. In essence there is no strand which is solely a guide strand
or solely a passenger strand because each strand serves as both
guide strand and passenger strand. The skilled artisan will also
appreciate that such molecules can be designed as single strands
with hairpin structures that can, for example, be processed in vivo
to become a duplex consisting of two separate strands.
[0142] In an embodiment, the invention provides a method for
designing a multitargeting interfering RNA molecule, comprising the
steps of: [0143] a) selecting one or more target RNA molecules,
wherein the modulation in expression of the target RNA molecules is
desired; [0144] b) obtaining at least one nucleotide sequence for
each of the target RNA molecules; [0145] c) selecting a length, n,
in nucleotides, for a seed sequence, wherein n=about 6 or more;
[0146] d) obtaining a collection of candidate seeds of the length n
from each nucleotide sequence obtained in step b), wherein a
candidate seed and its complete complement are not palindromic, and
the candidate seed occurs at least once in one or more of the
nucleotide sequences obtained in step b), and its complete
complement occurs at least once in one or more of the nucleotide
sequences obtained in step b); [0147] e) determining the genetic
context of each of the candidate seed and its complete complement,
by collecting, for each occurrence of the candidate seed and its
complete complement, a desired amount of the flanking sequences;
[0148] f) selecting a seed of the length n from the group of
candidate seeds; [0149] g) selecting a first consensus target
sequence, which comprises the seed and a consensus 3'-flanking
sequence to the seed determined from the sequences obtained in step
b); [0150] h) selecting a second consensus target sequence, which
comprises the complete complement of the seed and a consensus 3'
flanking sequence to the complete complement of the seed determined
from the sequences obtained in step b); [0151] i) obtaining a first
strand sequence, which comprises the first consensus target
sequence selected in step g) and, adjacent to and connected with
the 5'-end of the first consensus target sequence, a complement of
the consensus 3' flanking sequence of step h); [0152] j) obtaining
a second strand sequence which comprises the second consensus
target sequence selected in step h) and, adjacent to and connected
with the 5'-end of the second consensus target sequence, a
complement of the consensus 3' flanking sequence of step g), and;
[0153] k) designing a multitargeting interfering RNA molecule
comprising a first strand having the first strand sequence in step
i) and a second strand having the second strand sequence obtained
in step j).
[0154] The method further comprise repeating steps g) to k) for
each seed of length n selected from the group of candidate seeds in
step f).
[0155] The method further comprises the step of repeating steps c)
to k) for another desired seed length. In one embodiment, the first
scan through target sequences will begin with any seed length (e.g.
n=9) and subsequent rounds of searching will either increase or
decrease the seed length (e.g. based on the number of seeds
returned in previous scans). A person of ordinary skill in the art
will recognize that the number of candidate seeds will increase as
the length of the seed is decreased.
[0156] One skilled in the art will realize that finding a candidate
seed present in at least one of the selected RNA sequences and in
at least one complement of the selected RNA sequences, is an
alternative to finding the candidate seed and its complete
complement in the selected RNA sequences.
[0157] One skilled in the art will recognize that these design
steps may be performed in a different order to produce an
equivalent final product. Also, one skilled in the art will
recognize that some steps can be substituted with alternative
procedures that are broadly equivalent as shown in the Examples.
One skilled in the art will appreciate that the six elements of
Formula 1 (X, S, Y, X', S', Y') can be determined and assembled in
a number of ways.
[0158] Often the preference for designing a multitargeting
interfering RNA molecule of the invention involves: firstly,
identifying the seed and its complement, which occur in different
genetic contexts; secondly, determining XS and Y'S' so as to bind
to their respective target RNA sequences, and then determining XSY
and Y'S'X' wherein Y is the complement of Y' and X' is the
complement of X. As an example, XS may be determined as the
complement of the seed (equates to S) together with the complement
of a portion of the 3' flanking sequence of the seed (equates to
X). Similarly, Y'S' may be determined as the complement of the
complement of the seed (equates to S') together with the complement
of a portion of the 3' flanking sequence of the complement of the
seed (equates to Y'). In cases in which it is desired to target a
plurality of sequences, the plurality of 3' flanking sequences may
be examined to yield consensus 3' flanking sequences. X and/or Y'
can then be determined as the complements of these consensus 3'
flanking sequences. Further modifications can be made to the
molecule as described in this specification.
[0159] Preferred target RNA molecules are strategically selected
molecules, for example viral or host RNAs involved in disease
processes, viral genomes, particularly those of clinical
significance, and the like. A detailed discussion of target RNA is
provided above and applies equally to this and other aspects of the
invention, as if set out in its entirety here. The basis for the
selection of a target RNA molecule will be appreciated by those of
skill in the art. Preferred target RNAs are those involved in
diseases or disorders one wishes to control by the administration
of the multitargeting interfering RNA.
[0160] The step of obtaining the sequences for the selected target
is conducted by obtaining sequences from publicly available
sources, such as the databases provided by the National Center For
Biotechnology Information (NCBI) (through the National Institutes
of Health (NIH) in the United States), the European Molecular
Biology Laboratories (through the European Bioinformatics Institute
throughout Europe) available on the World-Wide Web, or proprietary
sources such as fee-based databases and the like. Sequences can
also be obtained by direct determination. This may be desirable
where a clinical isolate or an unknown gene is involved or of
interest, for example, in a disease process. Either complete or
incomplete sequences of a target RNA molecule can be used for the
design of multitargeting interfering RNA of the invention.
[0161] Also provided herein are methods wherein a plurality of
independent target nucleotide sequences are obtained in step b) for
each of one or more target RNA molecules selected in step a). The
databases described above frequently have multiple sequences
available for particular targets. This is especially true where
genetic variation is naturally higher, for example with viral
sequences. In various embodiments, the plurality of target
nucleotide sequences represents strain variation, allelic
variation, mutation, or multiple species. The number of such a
plurality of sequences may range from several or a low multiple, to
numerous--for example dozens or even hundreds or thousands of
sequences for a given target. It is especially possible to have
such numbers of sequences when working with viral sequences.
[0162] The sequences chosen can be further limited based on
additional desirable or undesirable features such as areas of low
sequence complexity, poor sequence quality, or those that contain
artifacts relating to cloning or sequencing such as inclusion of
vector-related sequences. Furthermore, regions with extensive
inaccessible secondary structure could be filtered out at this
stage. Indeed, Luo and Chang have demonstrated that siRNA targeting
accessible regions of mRNA structure such as loops were more likely
to be effective than those aligned with stems (Luo & Chang,
(2004), Biochem. Biophys. Res. Commun., 318: 303-10). The sequences
chosen, however, need not be limited to 3'UTR sequences or regions
of low secondary structure.
[0163] The step of selecting a length of n nucleotide bases for a
seed sequence is preferably an iterative process that does not
require any particular basis or logic at first glance--i.e. the
starting seed length may be any number of bases above about 6. The
longer the length that is chosen for a seed, the less likely that
it and its complete complement will appear in the at least one
target RNA, e.g. in a target RNA sequence. The shorter the seed
sequence length, the more frequently it will occur as would be
expected. Preferably, an iterative process is used to find the
preferred sequences for candidate seeds as described below. Thus,
after a particular value for n is used to identify candidate seeds
of length n, another value (e.g. n+1, n-1) will be used and the
process can be repeated to identify candidate seed sequences of
length n+1, n-1 and so on.
[0164] The seeds are selected from a pool of "candidate seeds,"
also referred to herein as "seed candidates." Seed candidates
include sequences of a particularly desired or selected length each
of which and its complete complement are not palindromic, and
wherein the candidate seed occurs at least once in one or more of
the nucleotide sequences obtained in step b), and its complete
complement occurs at least once in one or more of the nucleotide
sequences obtained in step b). The candidate seeds are preferably
generated by computer, for example by moving stepwise along a
target sequence with a "window" (expressed in terms of a fixed
number of contiguous nucleotides) of the desired or selected seed
length. Preferably each step is a single base progression, thus
generating a "moving window" of selected length through which each
target sequence is sequentially viewed. Other step distances are
contemplated, however, the skilled artisan will appreciate that
only a step of one nucleotide will allow the generation of all
possible seeds sequences.
[0165] Particularly, a collection of candidate seeds of the length
n can be obtained by the steps of: [0166] i) generating a first
collection of sequences of the length n from each of the nucleotide
sequences chosen for the target molecules, using a method
comprising the steps of: [0167] 1) beginning at a terminus of each
of the nucleotide sequence; [0168] 2) sequentially observing the
nucleotide sequence using a window size of n; and [0169] 3)
stepping along the nucleotide sequence with a step size of 1;
[0170] ii) generating a second collection of sequences each of
which is completely complementary to a sequence in the first
collection; and [0171] iii) obtaining the collection of candidate
seeds of the length n from the inspection of the first and the
second collections of sequences, wherein a candidate seed and its
complete complement are not palindromic, and each of the candidate
seed and its complete complement each occurs at least once in the
nucleotide sequences for the target molecules.
[0172] In another embodiment, a collection of candidate seeds of
the length n can be obtained by the steps of: [0173] i) obtaining
the completely complementary sequence for each nucleotide sequence
chosen for the target molecules; [0174] ii) generating a first
collection of sequences of the length n from each of the nucleotide
sequences chosen for the target molecules and a second collection
of sequences of the length n from each of the completely
complementary sequences obtained in step (i), using a method
comprising the steps of: [0175] 1) beginning at a terminus of the
nucleotide sequence of each of the nucleotide sequences chosen for
the target molecules or each of the completely complementary
sequences obtained in step (i); [0176] 2) sequentially observing
the nucleotide sequence using a window size of n; and [0177] 3)
stepping along the nucleotide sequence with a step size of 1; and
[0178] iii) obtaining the collection of candidate seeds of the
length n from the inspection of the first and the second
collections of sequences, wherein a candidate seed and its complete
complement are not palindromic, and each of the candidate seeds is
present in both the first and the second collections of
sequences.
[0179] In one embodiment, the method further comprises the step of
discarding candidate seed sequences for which either the seed or
its complete complement do not occur with at least a predetermined
minimum frequency in the target nucleotide sequences.
[0180] Preferably the method ultimately chosen will include one or
more of these steps, or all of them as needed. For example, in one
embodiment, the method further comprises the step of discarding any
candidate seed sequence that: is composed of only a single base, is
composed only of A and U, has a consecutive string of 5 or more C
or 5 or more G, is predicted to occur with unacceptable frequency
in the non-target transcriptome of interest; is predicted to have a
propensity to undesirably modulate the expression or activity of
one or more cellular component (eg. to undesirably activate a
cellular sensor of foreign nucleic acid), or any combination
thereof.
[0181] Seeds then are selected from the pool of candidate sequences
as the ones where the seed is present in one genetic context, and
its complete complement is present in a different genetic context
in the at least one pre-selected target sequence. Genetic contexts
are determined by collecting, for each occurrence of the candidate
seed sequence, a desired amount of the 5' and 3' flanking sequence.
The genetic context of the complement of the seed is determined in
a similar fashion.
[0182] In an exemplary process of making a multitargeting
interfering RNA of the invention when one or both strands are each
required to target a plurality of RNA sequences (e.g. multiple
viral isolates), a "consensus target sequence" is selected for one
or both strands of the interfering RNA.
[0183] The term "consensus target sequence" does not suggest that
there is only one best sequence approximating multiple binding
sequences on target molecule(s), rather a population of one or more
alternative sequences may all be consensus target sequences.
[0184] A first consensus target sequence for the first strand of
the iRNA comprises a seed sequence and a consensus 3'-flanking
sequence to the seed in at least one of the chosen sequences for
the target molecules. A second consensus target sequence for the
second strand of the iRNA comprises the complete complement of the
seed and a consensus 3' flanking sequence to the complete
complement of the seed in at least one of the chosen sequences for
the target molecules.
[0185] The "consensus 3' flanking sequence" of the seed is readily
derived by visual inspection, or through the use of bioinformatic
tools or calculations, from the examination of the genetic context
of each occurrence of the seed sequence in the sequences of the
target molecules. While the seed portion of the consensus target
sequence has complete identity to a corresponding portion in each
of the targeted binding sites, the consensus 3' flanking sequence
need not be completely identical, but can be identical, to the
sequence/s flanking the 3' end of the seed of one or more of the
target sequences. Likewise, the "consensus 3' flanking sequence" of
the complement of the seed is readily derived by visual inspection,
or through the use of bioinformatic tools or calculations, from the
examination of the genetic context of each occurrence of the
complement of the seed. While the complement of the seed portion of
the consensus target sequence has complete identity to a
corresponding portion in each of the targeted binding sites, the
consensus 3' flanking sequence need not be completely identical,
but can be identical, to the sequencers flanking the 3' end of the
complement of the seed.
[0186] Preferably, the consensus target sequence does not include
any sequence that is predicted to have a propensity to undesirably
modulate the expression or activity of one or more cellular
component.
[0187] Consensus target sequences may be determined by eye or by
algorithm. For example, a computer algorithm can be used to score
all possible permutations of paired nucleotides in the positions in
which the sequences are different. This is particularly useful when
the target sequences have some identity beyond the seed, but for
which an alignment by eye proves difficult. This method can be used
to determine the consensus target sequence/s, or alternatively,
directly design the strands of the candidate multitargeting
interfering RNA.
[0188] One alternative approach that is particularly useful when a
large number of target sequences need to be considered (e.g. when
large numbers of nucleotide sequences for viral isolates are
screened) is to generate all possible permutations of the extension
from the seed to a required length, and/or the complete complement
of the seed to a required length, thereby generating the putative
Y'S' and/or XS of Formula (I) and hybridizing each putative XS and
Y'S' against the target sequences of interest in silico to
determine those which demonstrate the most favorable properties in
terms of hybridization to the target.
[0189] Sequences demonstrating strong binding (typically having
mean free energies of <-20 kcal/mol) are of particular interest
for the multitargeting interfering RNA. Regardless of the flow path
of design, the candidate XS and Y'S' are then prioritized for
testing not only on this basis but also taking into account other
features that may be important for the functionality of the
multitargeting interfering RNA (by, for example, use of appropriate
penalty terms). This may involve discarding those putative XS and
Y'S' sequences which are composed of only a single base, are
composed only of A and U, are predicted to be involved in
substantial intramolecular base pairing, have a consecutive string
of 5 or more bases which are G, are predicted to occur with
unacceptable frequency in the antiparallel orientation in the
non-target transcriptome of interest; are predicted to have a
propensity to activate a cellular sensor of foreign nucleic acid,
or any combination thereof.
[0190] In some cases, the addition of one or two nucleotides to the
5' end of the putative XS or Y'S' that are not complementary to
their respective target sequences is considered. This is
particularly relevant when an otherwise useful XS or Y'S' is G/C
rich at the 5' end and this is predicted to disfavor loading
relative to the other strand. The addition of one or two A/U
nucleotides to the 5' extremity of the G/C rich XS or Y'S' will
most likely promote balanced loading, which is required for optimal
activity of the multitargeting RNA. Because multitargeting
interfering RNAs in most cases tolerate mismatches at positions 1
and 2, the addition of this additional region, which need not be
complementary to the corresponding target sequences, further
increases the flexibility of design. Finally, one skilled in the
art will appreciate that modifications that disfavor strand loading
could be used on the 5' end of the strand present at the
thermodynamically weaker end of the duplex to further enhance the
loading of the opposite strand. Such modifications also include
manipulation of the length and composition of the overhangs. Also,
the substitution of U for C in the corresponding strand will at
least partially rectify strand loading when there is a G near the
5' terminus of the XS or Y'S' by virtue of the wobble base pairing
present between U and G, which is weaker than the pairing between C
and G. Substitutions with chemically modified bases such as 2'F,
2'-O-methyl and LNA modified ribonucleotides increase the energy of
hybridization of nucleotides with matching bases. Therefore, the
alternative strategy of strengthening the hybridization of the
duplex at the thermodynamically weaker end with chemically modified
bases is also envisaged in this invention.
[0191] DNA sequences with stretches of contiguous guanosines are
known to produce additional effects not related to targeting of
mRNA. Although the situation in the case of RNA is less clear, most
manufacturers recommend not selecting dsRNA duplexes containing
long runs of G for their experiments. It was found in this
invention that greater than 4 consecutive G greatly reduced the
activity of the corresponding CODEMIR (data not shown). Therefore,
many seeds could be eliminated if a requirement for 5 or more C is
applied. One skilled in the art will recognize that the presence of
5 or more Cs in a seed will correspond to 5 or more Gs in the
completely complementary RNA molecule of the invention.
[0192] In another embodiment, the method further comprises the step
of discarding any consensus target sequence that: is composed of
only a single base, is composed only of A and U, has a consecutive
string of 5 or more bases which are C, is predicted to occur with
unacceptable frequency in the non-target transcriptome of interest,
is predicted to have a propensity to undesirably modulate the
expression or activity of one or more cellular component, or any
combination thereof.
[0193] Scanning the consensus target sequences against a
transcriptome of interest for prediction of off-target effects, and
eliminating any sequence predicted to have unacceptable off-target
effects on a transcriptome of interest are also useful ways of
reducing the number of consensus target sequences, and any of the
foregoing may be added as a step in the process. In practice, it is
prudent to routinely screen specific designed multitargeting
interfering RNAs, e.g. CODEMIRs, VIROMIRs and the like, for
cytotoxicity, due to unforeseen, but problematic, off-target
effects.
[0194] Any undesirable properties for a therapeutic RNA, as would
be understood by those of skill in the art, can be used as a basis
on which to discard candidate seed sequences, consensus binding
sites or proposed multitargeting interfering RNA.
[0195] Like candidate seeds and seeds, consensus target sequences
are intermediates in the design of a multitargeting interfering RNA
of the invention. In particular, the consensus target sequences are
used to generate the sequences for the first and the second strand
of a multitargeting interfering RNA of the invention. The first
strand sequence is designed to comprise the first consensus target
sequence and a complement of the consensus 3' flanking sequence of
the second consensus target sequence, which is adjacent to and
connected with the 5'-end of the first consensus target sequence.
In a particular embodiment, the first strand is designed by
extending the first consensus target sequence in the 5' direction
with a complete complement of consensus 3' flanking sequence of the
second consensus target sequence. The second strand sequence is
designed to comprise the second consensus target sequence and a
complement of the consensus 3' flanking sequence of the first
consensus target sequence, which is adjacent to and connected with
the 5'-end of the second consensus target sequence. In a particular
embodiment, the second strand is designed by extending the second
consensus target sequence in the 5' direction with a complete
complement of consensus 3' flanking sequence of the first consensus
target sequence.
[0196] In most cases, the overhangs, if required, are considered as
part of the hybridization process Hybridization is typically
examined from a thermodynamic perspective using RNAhybrid software
(Rehmsmeier et al., 2004, RNA, 10: 1507-17) or similar
algorithm.
[0197] In particular embodiments, X and Y' in Formula (I) are
completely complementary to their respective target sites. In the
case in which the X and Y', by virtue of being simply complementary
to their respective target sites result in very different G/C
richness at the two ends, then the loading bias needs to be reduced
by either producing mismatches in either X' or Y, depending on the
thermodynamic balance. Alternatively, several chemical
modifications (eg LNA, 2'O-methyl and 2'F can be introduced into
the "weak" end of the duplex to improve loading balance. Also, as
shown in the Examples, varying the length of the overhang may be
used to control the loading balance of the two strands of the
duplex.
[0198] It will be readily appreciated by one skilled in the art
that in the case of a double stranded multitargeting interfering
RNA of the invention, ensuring similar strand loading of both
strands is beneficial not only with respect to the potency of the
molecule but is also required to obtain multitargeting activity. In
a preferred embodiment, the multitargeting interfering RNA is
designed such that there is no loading bias, so that both strands
can load equally.
[0199] Various steps can optionally be added, individually or in
combination, to further the rational process of designing the
RNAs--such as to reduce the number of sequences unlikely to work
for the intended purpose, to increase the effectiveness of the
RNAs, to reduce off target effects and the like. Many of these
steps can be automated, or require only a limited amount of input
from an operator, though the use of bioinformatic computer systems,
which as the skilled artisan will appreciate, will facilitate the
methods.
[0200] Similar to the situation with antisense, for which it is now
recognized that there are specific sequences that have a high
propensity to activate cellular sensors of foreign DNA, other
receptors may detect particular RNA sequences and produce stress
responses (for example, see Sioud, M. (2005), J Mol Biol 348,
1079-1090. Specific "motifs" associated with increased inflammatory
responses (Hornung, V. et al. (2005) Nat Med 11, 263-270; Judge, A.
D. et al. (2005) Nat Med 11, 263-270) could be easily excluded.
[0201] In particular embodiments, the following sequences in the
context of Formula (I) are discarded: [0202] Occurrences of greater
than 4 contiguous Gs in XSY or Y'S'X' [0203] Greatly different G/C
frequency in the X and Y regions (by function of duplex, this means
also X' and Y') because loading bias is unlikely to be able to be
equalized. [0204] XS or Y'S' composed of only a single base or
composed only of A and U or predicted to occur with unacceptable
frequency in a non-target transcriptome of interest. [0205] Where
either of the strands are predicted to be involved in strong
intramolecular base pairing. [0206] Where the duplex comprises
elements predicted or known to have a propensity to activate a
cellular sensor of foreign nucleic acid.
[0207] In certain embodiments, the designed multitargeting
interfering RNA molecule can be modified, for example, i) to
improve actual or predicted loading of the strands of the
multitargeting interfering RNA molecule into the RNA induced
silencing complex (RISC); ii) to increase or decrease the
modulation of the expression of at least one target RNA molecule;
iii) to decrease stress or inflammatory response when the
multitargeting interfering RNA molecule is administered into a
subject; iv) to alter half-life in an expression system; or v) any
combination of i) to iv).
[0208] The skilled artisan will understand how to modify the RNA
molecules either in the laboratory, or preferably in silico. In
preferred embodiments the modifying step comprises one or more of
altering, deleting, or introducing one or more nucleotide bases to
create at least one mismatched base pair, wobble base pair, or
terminal overhang, or to increase RISC mediated processing.
Techniques for doing so are known in the art. Preferably the
modifications are at least initially performed in silico, and the
effects of such modifications can be readily tested against
experimental parameters to determine which offer improved
properties of the interfering RNA products.
[0209] Also provided herein are methods that further comprise the
step of actually making and testing at least one designed
interfering RNA in a suitable cellular expression system. This will
be necessary so as to identify those interfering RNA that have the
required or sufficient activity against the target RNA molecules or
that produce the required phenotype in the model system (eg death
of cancer cell, inhibition of angiogenesis, suppression of lesion
formation, accelerated wound healing etc).
[0210] In a presently preferred embodiment, the methods, through to
the step of actually making an RNA, are conducted entirely in
silico, or by visual inspection and determination. In one
embodiment the method further comprises the step of choosing a new
value for the seed length, n, and repeating each of the remaining
steps. It is clear that the method can be iterative and the
benefits of computers for such purposes are well known.
[0211] As will be appreciated, large numbers of seeds and thereby
potential multitargeting interfering RNAs can be generated using
the above methodology. While the rules above can be used to filter
potential candidates based on undesirable properties, one skilled
in the art will appreciate that with access to high throughput
screening methodologies as well as recent improvements in quality,
cost and access to RNA synthesis that testing of large numbers of
candidates can be easily performed to further assist in the
development of active multitargeting interfering RNAs. While the
testing of a number of multitargeting interfering RNAs may be
needed to identify those molecules with the greatest efficacy for a
desired application, those skilled in the art of molecular biology
will appreciate that this work does not amount to undue
experimentation. Thus, it is occasionally preferable to screen
significant numbers of candidates as opposed to prioritizing a few
candidates solely on the basis of algorithmic design. A combination
of careful in silico design along with biological testing of
candidates can be used to identify candidates with superior
activity in an efficient manner.
[0212] Screens that can be considered for the high throughput
assessment of candidates include reporter assays, multiplexed
ELISAs, viral replicon systems, dot-blot assays, RT-PCR etc.
[0213] Candidate multitargeting interfering RNA are routinely
synthesized as double-stranded RNA molecules with 19 bp of
complementarity and 3' two nucleotide overhangs. The overhang can
be any nucleotides or analogs thereof, such as, for example, dTdT
or UU. However, other types and lengths of overhangs could also be
considered, as could "blunt-ended" duplexes. In a preferred
embodiment, the overhangs are incorporated a priori into the design
by having Y and X' being longer than the corresponding X and Y' by
the length of the required overhangs.
[0214] When produced by an expression system such as a vector or
plasmid, it is possible to assemble multiple multitargeting
interfering RNAs into a single therapeutic product. Skilled
artisans will realize that multiple multitargeting interfering RNAs
can be co-expressed by several strategies, including but not
limited to, expression of individual multitargeting interfering
RNAs from multiple expression vectors (plasmid or viral),
expression from multiple expression cassettes contained within a
single vector, with each expression cassette containing a promoter,
a single multitargeting interfering RNA and terminator. Multiple
multitargeting interfering RNAs can also be generated through a
single polycistronic transcript, which contains a series of
multitargeting interfering RNAs.
[0215] The multitargeting interfering RNAs can be expressed
sequentially (sense/intervening loop/antisense) or expressed with
the sense sequence of each multitargeting interfering RNA
sequentially linked 5' to 3', joined directly or with intervening
loop/spacer sequence, followed by the antisense sequence of each
multitargeting interfering RNAs sequentially linked 5' to 3'.
[0216] In the first instance, multitargeting interfering RNA are
typically tested in cell culture using an appropriate cell line
representative of the targeted tissue. The specific conditions used
are outlined in the specific examples. Multitargeting interfering
RNA that lead to reduction in target RNA expression can then be
studied further. Specifically, semi-quantitative RT-PCR for the
target RNA may be performed to establish whether modulation of
expression of a target RNA is likely to be mediated by degradation.
In general, cells are transfected with the multitargeting
interfering RNA at a concentration of 5-40 nM in the culture medium
and after 48 hours, are washed, trypsinized and harvested for total
RNA using a RNeasy kit (Qiagen). RT-PCR is then performed using
primer sets specific for the target RNAs.
[0217] Proteomic and microarray experiments may be used to assess
off-target effects. Likewise, to select active multitargeting
interfering RNA with little propensity for activation of innate
immune response, analysis of markers of IFN-response (eg STAT1,
IFNb, IL-8, phosphoEIF etc) can be performed on treated cells.
[0218] Preferably, the candidate multitargeting interfering RNA are
tested for non-specific toxic effects by, for example, direct
assays of cell toxicity. Alternatively, in some cases such as
cancer, cytotoxicity is the desired outcome and may reflect the
successful repression of key oncogenic signaling pathways.
Multitargeting interfering RNA are additionally assessed for their
ability to repress the production of specific target proteins.
Multitargeting interfering RNA demonstrating efficacy in this
respect are then assessed for additional evidence of off-target
effects, including arrest of non-target protein production and
activation of Protein Kinase R (PKR) mediated responses.
[0219] The RNA molecule may be expressed from transcription units
inserted into vectors. The vector may be a recombinant DNA or RNA
vector, and includes DNA plasmids or viral vectors. The
multitargeting interfering RNA molecule expressing viral vectors
can be constructed based on, but not limited to, adeno-associated
virus, retrovirus, adenovirus, lentivirus or alphavirus.
[0220] Preferably the vector is an expression vector suitable for
expression in a mammalian cell.
[0221] Methods which are well known to those skilled in the art can
be used to construct expression vectors containing a sequence which
encodes the multitargeting interfering RNA molecule. These methods
include in vitro recombinant DNA techniques, synthetic techniques
and in vivo recombination or genetic recombination. Such techniques
are described in Sambrook et al (1989) Molecular Cloning, A
laboratory manual, Cold Spring Harbor Press, Plainview N.Y. and
Asubel F M et al (1989) Current Protocols in Molecular Biology,
John Wiley & Sons, New York N.Y. Suitable routes of
administration of the pharmaceutical composition of the present
invention may, for example, include oral, rectal, transmucosal, or
intestinal administration; parenteral delivery, including
intramuscular, intravenous and subcutaneous injections.
[0222] Alternatively, the pharmaceutical composition may be
administered in a local rather than systemic manner, for example,
via injection of the pharmaceutical composition directly into a
target organ or tissue, such as intramedullary, intrathecal, direct
intraventricular, intraperitoneal, or intraocular injections, often
in a depot or sustained release formulation. Intravesicular
instillation and intranasal/inhalation delivery are other possible
means of local administration as is direct application to the skin
or affected area. Ex vivo applications are also envisaged.
[0223] Furthermore, the pharmaceutical composition of the present
invention may be delivered in a targeted delivery system, for
example, in a liposome coated with target cell-specific antibody.
The liposomes will be targeted to and taken up selectively by the
target cell. Other delivery strategies include, but are not limited
to, dendrimers, polymers, nanoparticles and ligand conjugates of
the RNA.
[0224] The multitargeting interfering RNA molecule of the invention
are added directly, or can be complexed with cationic lipids,
packaged within liposomes, or otherwise delivered to target cells
or tissues. The nucleic acid or nucleic acid complexes can be
locally administered to relevant tissues ex vivo, or in vivo
through injection, infusion pump or stent, with or without their
incorporation in biopolymers.
[0225] In another aspect, the invention provides biological systems
containing one or more multitargeting interfering RNA molecule of
this invention. The biological system can be, for example, a virus,
a microbe, a plant, an animal, or a cell. The invention also
provides a vector comprising a nucleotide sequence that encodes the
multitargeting interfering RNA molecule of the invention. In
particular embodiment, the vector is viral, for example, derived
from a virus selected from the group consisting of an
adeno-associated virus, a retrovirus, an adenovirus, a lentivirus,
and an alphavirus. The multitargeting interfering RNA can be a
short hairpin RNA molecule, which can be expressed from a vector of
the invention. The invention further provides a pharmaceutical
composition comprising a multitargeting interfering RNA molecule of
the invention and an acceptable carrier. In particular embodiments,
the pharmaceutical composition comprises a vector for a
multitargeting interfering RNA molecule of the invention.
[0226] In another general aspect, the present invention provides a
method of inducing RNA interference in a biological system, which
comprises the step of introducing a multitargeting interfering RNA
molecule of the invention into the biological system.
[0227] The invention further comprises a method of treating a
subject, comprising the step of administering to said subject a
therapeutically effective amount of a pharmaceutical composition
comprising a multitargeting interfering RNA molecule of the
invention. The invention also provides a method of inhibiting the
onset of a disease or condition in a subject, comprising
administering to said subject a prophylactically effective amount
of a pharmaceutical composition comprising a multitargeting
interfering RNA molecule of the invention. Methods are known in the
art for determining therapeutically and prophylactically effective
doses for the instant pharmaceutical composition.
[0228] The compositions and methods exemplified herein are of use
in the treatment of complex multigenic diseases in which single
gene-specific therapeutics may be at a disadvantage because of the
multiple redundancies in pathophysiologic signaling pathways. A
conscious and calculated approach is provided in which key
signaling proteins/pathways can be simultaneously targeted with a
single agent to generate greatly increased therapeutic
potential.
[0229] In some cases, the targets of interest may be at least
partially controlled by a common "master regulator". This is
usually a transcription factor. For example, down-regulation of
IL-8 and MCP-1 might be achievable through targeting the nuclear
factor NFkappaB. However, as an example, this pathway is also
involved in the survival of Retinal Pigmented Epithelial cells
(RPE) in times of stress and the down regulation of such a
cell-survival factor would likely lead to increased death of RPE in
diseased eyes. Therefore, the novel approach disclosed herein has
the advantage of being amenable to the modulation of specific
targets of interest without having to identify suitable target
"upstream" pleiotropic controllers.
[0230] Application also exists in the treatment of diseases
characterized by cellular heterogeneity. For example, in solid
tumours, the presence of mutated genes and activated pathways may
vary widely within the same tumour, between tumours in the same
patient as well as between tumours of a similar histology in
different patients. In such instances, the development of an RNA
molecule active against several key pathways may derive synergistic
activity against cells reliant on several of these targeted
pathways. However, activity against a greater proportion of the
tumour cells will also be likely because of the "multi-targeted"
nature of the RNA molecule of the invention. Furthermore, targeting
of several key pathways will "cover" more of the patient
population. Hence, improved clinical outcomes are likely with
treatment with the RNA molecules exemplified or taught herein.
[0231] In cases in which RISC is involved in the mechanism of
action, the targeting of multiple disease-related transcripts with
a single multitargeting interfering RNA molecule of the invention
(eg a CODEMIR or VIROMIR) preferably allows full use of available
RISC as opposed to the administration of multiple siRNA molecules,
which could, in some cases, saturate the available intracellular
machinery.
[0232] Targeting multiple sites within the same RNA target sequence
can also be accomplished with the compositions and methods provided
herein. Many human diseases, including cancer and viral infections,
are characterized by RNA targets exhibiting high mutation rates.
This increases the likelihood of resistance to nucleic acid
therapeutics arising in these diseases, due to mutation of the
target RNA. Targeting multiple sites within the target RNA
decreases the likelihood of such resistance arising, since at least
two simultaneous mutations would be required for resistance. In
this instance, therefore, the multi-targeting approach of
multitargeting interfering RNAs (eg CODEMIRs or VIROMIRs) is
directed to the generation of multiple hits against a single target
RNA to prevent escape mutants. Targeting of multiple sites within
the same transcript (eg. as in the case of RNA viruses) may also
produce synergistic effects on the inhibition of viral growth.
Further, employing a mechanism or mechanisms requiring only partial
complementarity with the target RNA can have an advantage in
decreasing the possibility of developing resistance through point
mutation.
[0233] The desired targets for any disease entity may be identified
based on an approach or a mixture of approaches including, but not
limited to, validated drug targets from literature and proprietary
target discovery processes. The target genes are then prioritized
based on evidence supporting a key role for their products in the
disease process of interest.
[0234] In some cases, specific attention may need to be paid to the
accuracy and/or relevance of the sequence to the disease of
interest. For example, in targeting cancer, it is advisable to
avoid mutational "hot-spots". Also, in some embodiments, selective
targeting of a specific splice variant or isoform may be desired
and thus in such embodiments, the target sequence used in
multitargeting interfering RNA design is preferably restricted to
that isoform or variant present only in diseased tissue.
[0235] The sequences of the target RNA or RNAs are preferably
downloaded from public or proprietary databases or generated from
sequencing experiments.
EXAMPLE 1
CODEMIRs to VEGF-A and ICAM-1
[0236] Approaches for the design of multi-targeting of ICAM-1 and
VEGF-A with CODEMIRs were considered.
[0237] The entire mRNA sequence for each of VEGF-A and ICAM-1 was
used. These sequences were searched to find sequences that are
present in the coding strand for one target and the complement of
the coding strand for another target. Here, the sequence
5'-AGTGACTGTCAC-3' (SEQ ID NO: 1) was identified both in the ICAM-1
coding sequence and in the complement of the VEGF-A coding
sequence. This sequence was used to design a CODEMIR active against
multiple targets, using each strand of the CODEMIR to target at
least one of the target RNAs. The sequence identified above and its
complement were used as a centrally-located part of a CODEMIR
duplex. Each strand of this central duplex was extended in the 5'
direction to provide full complementarity to the corresponding
target, whereas each strand was extended in the 3' direction so as
to be complementary to its opposing strand in the CODEMIR duplex
strand.
[0238] The skilled artisan will appreciate that rationally
designing CODEMIRs requires designing portions of both strands (as
a duplex) together and gradually lengthening the duplex, and
refining to complete the design. The rational design process can
continue after the duplex is largely complete--as the refinements
may be made to modify the ends, or to create mismatches or wobble
pairings to improve loading or other aspects of functionality.
[0239] The length of this central region (12 nt) leads to two
possible 21-base duplexes with 3' double overhangs depending on how
the remaining required sequence is divided into the two sequences
surrounding the seed. Each of those CODEMIRs is shown in Table 1-1
(CODEMIRs-16 and -17).
[0240] It will be appreciated by one skilled in the art that these
CODEMIRs comprise a sequence that is the complement of the seed. In
this example, the complementary sequence in the CODEMIRs is
GUGACAGUCACU. (SEQ ID NO: 2)
[0241] CODEMIRs-16 and -17 were tested for activity against both
VEGF-A and ICAM-1 targets in RPE cells. RPE cells in culture were
used to screen the anti-angiogenic CODEMIRs designed, as described
above. The human cell line, ARPE-19, was used. ARPE-19 cells were
grown in Dulbecco's Modified Eagle's Medium supplemented with 10%
fetal bovine serum and 10 mM glutamine. For ELISA detection of
secreted proteins of interest, or in situ cell surface antigen
immunostaining, ARPE cells were seeded at 4.times.10.sup.3 cells
per well in a 96 well tissue culture plate. For FACS analysis,
ARPE-19 cells were seeded at 2.5.times.10.sup.4 cells per well in a
24 well tissue culture plate. Cells were transfected 24 hours after
seeding using lipofectamine (InVitrogen) at a ratio of 1 microL
lipofectamine per 20 .mu.mol of CODEMIR RNA duplex or control
siRNA. In most studies, medium was replaced 24 hours after
transfection at which time deferoxamine (130 .mu.M) or IL-1.beta.(1
ng/mL) was added for the VEGF-A and ICAM-1 experiments,
respectively. Experiments were performed in triplicate and repeated
at least twice.
[0242] The ARPE-19 cells were assayed to confirm production of both
VEGF-A and ICAM-1. VEGF-A was assayed in the supernatant using a
commercially available ELISA assay (R&D Systems) according to
the manufacturer's instructions. Cell surface ICAM-1 was assayed
either by immunostaining followed by fluorescence activated cell
sorting (FACS), by in situ immunostaining of cell-surface ICAM-1 in
96 well plates using colorimetric detection, or alternatively by
ELISA of cell lysates using a commercial sICAM ELISA kit (R&D
systems).
[0243] The results are shown in FIG. 1. Both CODEMIRs were active
modulators of the multiple targets. CODEMIR-16 was more active in
modulating VEGF-A and CODEMIR-17 was more active against ICAM-1,
apparently as a result of the design symmetry. This is likely due
to altered strand-loading bias.
[0244] The loading bias can be adjusted, for example, by
introducing wobble G:U basepairs into the extremities of the
duplex, or by expanding the CODEMIR to a 22-base duplex with
symmetrical extremities. Variations of each of these types were
explored. CODEMIR-26 is a 22-base duplex that has 4 identical
binding nucleotide pairs at each of the two termini of the duplex.
As shown in FIG. 1, CODEMIR-26 exhibited greatly increased ICAM-1
targeting compared to that of CODEMIR-16. Thus, the adjustments to
the sequence were able to correct the loading bias observed with
CODEMIR-16.
[0245] CODEMIRs-27 and -28 (see Table 1-1) were designed to test
whether disrupting strong G:C pairs at an end of the duplex region
would also successfully overcome the loading bias observed with
CODEMIR16. As can be seen from the results in FIG. 1, the
substitution of a C with a U in the 3'terminal region of the guide
strands targeting VEGF-A was successful in changing the bias (e.g.
CODEMIR-27). CODEMIR-28 had similar activity to CODEMIR-27 where
changes were made at the other end of the CODEMIR.
[0246] It was also envisaged herein that both strand loading bias
and target activity can be controlled by introducing mismatches to
disrupt the end of the duplex that is inefficiently loaded and
simultaneously increase hybridization to the target. For example,
in CODEMIR-36, a variant of CODEMIR-16, both strands were designed
to be entirely complementary to the respective target sequences;
the resulting incompletely complementary duplex features several
mismatches at the 5'extremity of the ICAM-1 guide sequence. The
results for CODEMIR-36 (see Table 1-1) are shown in FIG. 1.
[0247] The multitargeting interfering RNA (CODEMIRs) herein
disclosed would be expected to be effective in multiple angiogenic
diseases of the eye. This is because secreted VEGF-A plays a major
role in all of these diseases (Witmer et al (2003) Prog Retin Eye
Res, 22, 1-29), although ICAM-1 overexpression may be an early
initiating event, particularly for diabetic retinopathy and macular
edema (Funatsu et al., (2005)Opthalmology, 112, 806-16.; Joussen et
al. (2002) Am J Pathol, 160, 501-9; Lu et al. (1999) Invest
Opthalmol V is Sci, 40, 1808-12. We have shown that several
CODEMIRs are able to suppress both VEGF-A and ICAM-1 production by
human retinal epithelium cells (ARPE-19 cell line). These cells are
a major contributor to the production of these proteins in these
ocular angiogenic diseases (Matsuoka et al., (2004) Br J Opthalmol,
88, 809-15, Yeh et al. (2004), Invest Opthalmol V is Sci, 45,
2368-73). RPE cells are also the primary site of uptake of foreign
nucleic acids in the eye and, for these two reasons, are the
appropriate cell model for evaluation of anti-angiogenic CODEMIRs
in opthalmology. The in vivo activities of two oligonucleotide
drugs correlated with their activity against RPE cells in culture
(Garrett et al. (2001) J Gene Med, 3, 373-83; Rakoczy et al.
(1996), Antisense Nucleic Acid Drug Dev, 6, 207-13) demonstrating
the value of this cell culture model. An advantage of this cell
line is that it forms polarized monolayers that mimic the RPE layer
of the eye (Dunn et al., (1996), Exp Eye Res, 62, 155-69).
TABLE-US-00002 TABLE 1-1 Design of CODEMIRs for the targeting of
VEGF-A and ICAM-1 Top strand 5' to 3' Bottom strand 3' to 5' VEGF
binding* ICAM binding* CODEMIR16 ICAM Guide CGAGUGACAGUCACUAGCUCC
5' GAUCG GUGACAGUCACUAGCUU 3' 5' CGGGGAAUCAGUGACUGUCACUCGA 3' (SEQ
ID NO: 3) (SEQ ID NO: 5) (SEQ ID NO: 6) UAGCUCACUGUCAGUGAUCGA 3'
UAGCUCACUGUCAGUGAUCGA 5' 3' CCUCGAUCACUGACAGUGAGC 5' (SEQ ID NO: 4)
(SEQ ID NO: 4) (SEQ ID NO: 3) VEGF Guide CODEMIR17 ICAM Guide
UCGAGUGACAGUCACUAGCUC 5' UGAUCG GUGACAGUCACUAGCU 3' 5'
GGGGAAUCAGUGACUGUCACUCGAG 3' (SEQ ID NO: 7) (SEQ ID NO: 9) (SEQ ID
NO: 10) CUAGCUCACUGUCAGUGAUCG 3' CUAGCUCACUGUCAGUGAUCG 5' 3'
CUCGAUCACUGACAGUGAGCU 5' (SEQ ID NO: 8) (SEQ ID NO: 8) (SEQ ID NO:
7) VEGF Guide CODEMIR26 ICAM Guide UCGAGUGACAGUCACUAGCUCC 5' UGAUCG
GUGACAGUCACUAGCUU 3' 5' CGGGGAAUCAGUGACUGUCACUCGAG 3' (SEQ ID NO:
11) (SEQ ID NO: 67) (SEQ ID NO: 13) CUAGCUCACUGUCAGUGAUCGA 3'
CUAGCUCACUGUCAGUGAUCGA 5' 3' CCUCGAUCACUGACAGUGAGCU 5' (SEQ ID NO:
12) (SEQ ID NO: 12) (SEQ ID NO: 11) VEGF Guide CODEMIR27 ICAM Guide
CGAGUGACAGUCACUAGCUCC 5' GAUCG GUGACAGUCAGUAGCUU 3' 5'
CGGGAAUCAGUGACUGUCACUCGA 3' (SEQ ID NO: 3) (SEQ ID NO: 5) (SEQ ID
NO: 6) UAGUUCACUGUCAGUGAUCGA 3' UAGUUCACUGUCAGUGAUCGA 5' 3'
CCCUCGAUCACUGACAGUGAGC 5' (SEQ ID NO: 14) (SEQ ID NO: 14) (SEQ ID
NO: 3) VEGF Guide CODEMIR28 ICAM Guide UCGAGUGACAGUCACUAGUUC 5'
UGAUCG GUGACAGUCACUAGCU 3' 5' GGGGAAUCAGUGACUGUCACUCGAG 3' (SEQ ID
NO: 15) (SEQ ID NO: 9) (SEQ ID NO: 10) CUAGCUCACUGUCAGUGAUCG 3'
CUAGCUCACUGUCAGUGAUCG 5' 3' CUUGAUCACUGACAGUGAGCU 5' (SEQ ID NO: 8)
(SEQ ID NO: 8) (SEQ ID NO: 15) VEGF Guide CODEMIR36 ICAM Guide
CGAGUGACAGUCACUGAUUCC 5' UGAUCGGUGACAGUCACUAGCUU 3' 5'
GGGAAUCAGUGACUGUCACUCGA 3' (SEQ ID NO: 16) (SEQ ID NO: 67) (SEQ ID
NO: 18) CUAGCCACUGUCAGUGAUCGA 3' CUAGCCACUGUCAGUGAUCGA 5' 3'
CCUUAGUCACUGACAGUGAGC 5' (SEQ ID NO: 17) (SEQ ID NO: 17) (SEQ ID
NO: 16) VEGF Guide
[0248] *Upper strand mRNA target, lower strand .dbd.CODEMIR guide
strand. The bold sequence identifies the sequence present in ICAM-1
and as its complement in VEGF-A around which the CODEMIR duplexes
were designed.
EXAMPLE 2
CODEMIRs to Gluc6P and Inppl1
[0249] CODEMIRs may also be suitable for the treatment of complex
metabolic diseases such as type 2 diabetes. Two potential gene
targets for the treatment of this disease are glucose-6-phosphatase
and Inppl1. Full transcript sequences were examined. Candidate
CODEMIRs from the best contiguous region of identity are shown for
each case in Table 2-1.
[0250] Regions of complementarity between the two targets were
found and the two identified seeds (Table 2-1: CUGCCUCGCCCAG (SEQ
ID NO: 19) and CUCCACAUCCAC) (SEQ ID NO: 20) were used as the
central motifs for two possible CODEMIR duplexes. The latter seed
and its complement were extended at their 5' ends to generate
duplexes in which each strand has 5' complementarity to one of the
target sequences (FIG. 2A). This is important because both strands
of the CODEMIR duplex are effectors and an increased region of
identity of each strand with its target needs to be extended to the
5' terminus, whereas the less critical 3' end requires less
complementarity (FIG. 2A). Some modification of the CODEMIRs can be
performed to tune the hybridization of the CODEMIR duplex, thereby
affecting the loading bias. Introduction of mismatches is one way
of achieving this (for example see: Ohnishi et al. (2005) Biochem
Biophys Res Commun. 329:516-21) and these mismatches can be chosen
also for their ability to increase binding of the 3' region of the
effector strands to their respective target transcripts (FIG. 2B).
In this situation, the CODEMIR duplex is then no longer composed of
two strands with complete complementarity, similarly to
CODEMIR-36.
[0251] It will be appreciated by one skilled in the art that
multitargeting interfering RNA molecules (CODEMIRs) will comprise
the sequence corresponding to the complement of the seed. In this
example, these complementary sequences are CUGGGCGAGGCAG (SEQ ID
NO: 21) and GUGGAUGUGGAG. (SEQ ID NO: 22) TABLE-US-00003 TABLE 2-1
Target sequences aligned with CODEMIRs for targeting Gluc6p and
Inppl1. Sequences Gluc6p 5' GUGUCAUCCCCUACUGCCUCGCCCAGGUCCUGGGCCAGC
3' (SEQ ID NO: 23) Inppl1 (complement) 5'
CAGGCACUCAUGCCUGCCUCGCCCAGCCCGCUGGCCCGC 3' (SEQ ID NO: 24)
Candidate CODEMIR duplex 1 (e.g. central duplex 1) Inppl1 targeting
strand 5' AUGCCUGCCUCGCCCAGGUCC 3' (SEQ ID NO: 25) Gluc6p targeting
strand 3' UACGGACGGAGCGGGUCCAGG 5' (SEQ ID NO: 26) Gluc6p 5'
GAUUCUUCCACUGGCUCCACAUCCACCCCACUGGAUCUUCA 3' (SEQ ID NO: 27) Inppl1
(complement) 5' ACCAGCCGCCCACCCUCCACAUCCACGCUCAGCGUGAACUU 3' (SEQ
ID NO: 28) Candidate CODEMIR duplex 2 (e.g. central duplex 2)
Inppl1 targeting strand 5' CACCCUCCACAUCCACCCCAC 3' (SEQ ID NO: 29)
Gluc6p targeting strand 3' GUGGGAGGUGUAGGUGGGGUG 5' (SEQ ID NO:
30)
EXAMPLE 3
VIROMIRs Targeting Multiple Sites Within the HIV Genome
[0252] The invention can be used to target proteins of interest
that are likely to be mutated in chronic forms of disease.
Mutations may be particularly prevalent in cancer and viral disease
in which drug-resistant forms often evolve. In this example,
VIROMIRs were designed to target multiple sites in the Human
Immunodeficiency Virus (HIV). The requirement for simultaneous
mutation at several sites, in order to overcome the effects of such
a VIROMIR, is likely to provide a high genetic hurdle to the
emergence of resistant viral clones or quasispecies. The genome of
the HXB2 strain of HIV I serotype B (GenBank Accession K03455) was
used as the principal sequence of interest and was examined with
bioinformatics methods detailed elsewhere in this application to
find seeds occurring at more than one location. All HIV I clade B
isolates in the LANL database as of 1 Aug. 2005 which contain full
sequences for any of the GAG, ENV, POL, TAT, VIF, VPR, VPU and NEF
genes were used in these analyses.
[0253] A 17-base seed and its complete complement were found in the
HIV reference strain genome as shown below: TABLE-US-00004 (SEQ ID
NO: 31) TCTAATTCCAATAATTCTTGTTCATTCTTTTCTTGCTGGTTTTGCGATTC
TTCAATTAAGGAGTGTATTAAGCTTGTGTAATT K03455r (SEQ ID NO: 32)
CTTTGAGCCAATTCCCATACATTATTGTGCCCCGGCTGGTTTTGCGATTC
TAAAATGTAATAATAAGACGTTCAATGGAACAG K03455
[0254] wherein, K03455r is a partial sequence of the complement of
the reference strain genome.
[0255] In the population of Clade .beta. isolates described above,
the seed (GCTGGTTTTGCGATTCT) (SEQ ID NO: 33) was found in 76% of
isolates, whereas its complement (AGAATCGCAAAACCAGC) (SEQ ID NO:
34) was found in 4% of isolates respectively.
[0256] Ultimately, an effective RNA therapeutic of the invention
should provide broad coverage of the affected population and it is
obviously desirable to target sequences that are highly represented
in this patient population. Therefore, the seed presented above
might not cover a sufficient proportion of the population.
Nevertheless, due to its unique size it was considered further in
the exemplification of the invention.
[0257] However, if conservation needed to be improved for this
seed, one skilled in the art would appreciate that using a
sub-segment of this seed could result in improved conservation. For
example, when two bases are removed, as in the following:
TABLE-US-00005 5' TGGTTTTGCGATTCT 3' (forward) (SEQ ID NO: 35) 5'
AGAATCGCAAAACCA 3' (reverse) (SEQ ID NO: 36)
the forward conservation remains at 76%, while the reverse
conservation increases substantially to 35%.
[0258] In order to prioritize and test candidate VIROMIRs, it is
important to have screening methods that are compatible with the
intended target sequence. The pNL4.3 assay is widely used in the
field of HIV research as a valuable, validated screen for drugs
active in HIV and was used by us to test candidate VIROMIRs.
However, there are some differences between the sequences of the
HIV component of the pNL4.3 plasmid and that of the reference HIV
strain (K03455) used in the design of the VIROMIR. Therefore,
comparison of the sequence of the reference strain and the sequence
of the pNL4.3 plasmid was carried out to confirm that the
above-mentioned VIROMIR was targeting a sequence also present in
the testing system. Other testing systems such as viral challenge
assays, fusion reporters, viral pseudoparticles among others, each
representing any multitude of therapeutically relevant or
irrelevant sequences could equally be considered.
[0259] An example of a VIROMIR duplex (VM011) targeting these two
seed sites is: TABLE-US-00006 5' UGCTGGUUUUGCGAUUCUAAA 3' (SEQ ID
NO: 37) 3' GAACGACCAAAACGCUAAGAU 5' (SEQ ID NO: 38)
Analyzing the ends of the duplex, it will be apparent to one
skilled in the art that the two strands would be unlikely to load
equally, given the greater G/C content at one end relative to the
other, therefore it would likely benefit from further optimization
as discussed elsewhere in this invention.
[0260] HIV generally causes chronic infection with in vivo viral
reservoirs. Consequently, VIROMIRs targeting HIV are most likely to
be therapeutically effective as cell-expressed short hairpin RNAs
(shRNAs) rather than as synthetic RNA duplexes because of a need
for continued therapeutic cover to prevent re-emergence from latent
sites.
[0261] The sequences for VM011 were used in the design of an shRNA.
Contiguous DNA sequences corresponding to: BamHI restriction site,
G initiator, VIROMIR passenger, Xho loop sequence (ACTCGAGA),
VIROMIR guide strand, polIII terminator and HindIII restriction
site were assembled and prepared as dsDNA. They were then cloned
into a pSIL vector under the control of a H1 promoter. The
resulting double-stranded DNA insert designed to encode an shRNA
VIROMIR approximating VM011 is shown below (loop sequence in
parentheses and terminator italicized): TABLE-US-00007 (SEQ ID NO:
39) 5' GATCC GCTTGCTGGTTTTGCGATTCTA (ACTCGAGA) TAGAATC
GCAAAACCAGCAAG TTTTTTGGA A (SEQ ID NO: 40) G CGAACGACCAAAACGCTAAGAT
(TGAGCTCT) ATCTTAGCGTTTTG GTCGTTC AAAAAACCT TTCGA- 5'
[0262] One skilled in the art will appreciate that when
transcribed, the encoded RNA folds into a hairpin structure, which
is modified by the cellular Drosha and Dicer proteins to generate
active VIROMIR RNA duplex(es). The skilled artisan will also
recognize that a number of variations of the design of the shRNA
construct could be considered. These include but are not limited
to: length, sequence and orientation of the shRNA duplex components
(guide strand, passenger strand, precursors), length and sequence
of the loop, choice of promoter, initiator and terminator sequences
as well as the cloning strategies used to assemble the final
construct.
[0263] This shRNA contruct was tested in HEK-293 cells by
co-transfecting with the pNL4.3 plasmid. Specifically, HEK-293
cells were seeded at density of 2.times.10 5 cells in 1 ml Optimem
medium/well in a 12-well plate. Cells were transfected 24 hr later
with 200 .mu.L DNA: Lipofectamine mix (200 ng pNL4.3 plasmid, 67 ng
VIROMIR pSIL construct in 100 .mu.L complexed with 2.7 .mu.L
Lipofectamine 2000 in Optimem). After changing the medium 24 hours
later, the production of p24 was assayed by collection of the
supernatant after a further 24 hours of incubation.
[0264] The production of p24 was expressed as a percentage of the
production from cells transfected with the empty control plasmid.
VM011 did not have any appreciable activity in this assay (data not
shown), perhaps reflecting the lack of equivalent loading, as
predicted from the analysis of the ends of the duplex.
Nevertheless, one skilled in the art will appreciate that other
design strategies (eg. alternative extension of the strands from
the seed and its complement, or variation of the shRNA construct as
described above) could be considered for the development of an
active VIROMIR of this design, which when combined with appropriate
screens such as the one used above, could be used to identify
useful therapeutics.
EXAMPLE 4
Dual Targeting of HCV and TNFa
[0265] In some cases of infectious diseases, multitargeting
interfering RNAs can be utilized to target both the genome of the
infectious agent and one or more key host "drivers" of the disease.
For example, TNF-alpha is considered a major disease-associated
factor in Hepatitis C Virus infection and its sequelae.
Investigation of the genome of HCV and the TNF-alpha mRNA was
undertaken.
[0266] On searching for 9 base seeds and their complete complements
in HCV and TNFa, the following seed of interest was one of several
identified: 5' ACTCCCCTG 3' (SEQ ID NO: 41)
[0267] This seed was selected because it is present in the HCV
genome with a conservation of 94% in 155 isolates of genotypes 1a
and 1b. This seed is actually present in two sites in HCV. We then
considered the nucleotides in the 3' direction from this seed to
establish which site should be primarily considered in the design
of the duplex. The extended sequences for the sites (+6 bases to
the 3' end) were as follows: TABLE-US-00008 ACTCCCCTGTGAGGA (site
#1) (SEQ ID NO: 42) ACTCCCCTGACGCCG (site #2) (SEQ ID NO: 43)
[0268] It was found that the rate of occurrence of the seed at the
first first site was much higher than at the second (92% vs 7%).
Therefore the further design of the multitargeting interfering RNA
was performed only considering the sequence of the 1.sup.st
site.
[0269] In genetic context terms, the seed is in the 5'NTR of HCV
and 3'UTR of TNFalpha. Shown below is the location of the seed in
the HCV sequence and in the antiparallel sequence for TNFalpha:
TABLE-US-00009 (SEQ ID NO: 44) 5' TGATGGGGGCGACACTCCACCATGAATC
ACTCCCCTG TGAGGAAC TACTGTCTTCACGCAGAAAGC 3' HCV (SEQ ID NO: 45) 3'
ACTGAATAGTAGGGCGATTACAGACACA ACTCCCCTG GGGAGCAG
AGGCTCAGCAATGAGTGACAG 5' TNFar
[0270] Because the seed is 9mer, the required extension for the
duplex of the required length (eg 19) is, for example, 5 bases in
the 3' direction of the target and 5 bases in the 5' direction as
the seed is usually in the middle of the double stranded duplex
(excluding the overhangs). By putting it in the middle, the
resulting two strands will have an equivalent portion of complete
complementarity with their respective targets when the process
outlined below is followed. This should ensure that binding of the
resulting two strands should be comparable. With a seed of, for
example, ten nucleotides, the extension by 5 on each side would
create a duplex of 20, whereas extension of 4 plus 5 or 5 plus 4
would yield a duplex of 19 nucleotides. Other permutations are
equally permissible depending on the lengths of the seed and the
desired duplex.
[0271] In this example, a means of generating one of the strands of
the duplex is as follows: [0272] Starting with the complement of
the seed of the target in the normal orientation (i.e. HCV) gives
the following sequence 5' CAGGGGAGU 3'. (SEQ ID NO: 46) [0273] Then
extend this sequence further in its 5' direction by taking the
complement of the next 5 bases of the HCV sequence at the 3' end of
the seed--this gives 5' CCUCACAGGGGAGU3'. (SEQ ID NO: 47) [0274]
This is followed by the addition of the complement of the next 5
bases of the second target which is in the antiparallel orientation
(i.e. TNFa) to give the first strand sequence of
5'-CCUCACAGGGGAGUUGUGU-3' (SEQ ID NO: 48)
[0275] The opposite strand (TNFa) is then the complement of the
first guide strand: 5'-ACACAACUCCCCUGUGAGG-3' (SEQ ID NO: 49) such
that the duplex is: TABLE-US-00010 5'-CCUCACAGGGGAGUUGUGU-3' (SEQ
ID NO: 48) 3'-GGAGUGUCCCCUCAACACA-5' (SEQ ID NO: 49)
[0276] The two guide strands have predicted binding to the two
targets of: TABLE-US-00011 HCV 5' GACACUCCACCAUGAAUCACUCCCCUGUGAGGA
3' (SEQ ID NO: 50) Guide#1 3' UGUGUUGAGGGGACACUCC 5' (SEQ ID NO:
48) mfe: -35.0 kcal/mol TNFa 5' GCCUCUGCUCCCCAGGGGAGUUGUGUC 3' (SEQ
ID NO: 51) Guide#2 3' GGGAGUGUCCCCUCAACACA 5' (SEQ ID NO: 49) mfe:
-35.0 kcal/mol
[0277] To improve equality of loading, the duplex could be extended
with further complementarity to the HCV sequence, possibly:
TABLE-US-00012 5'-UCCUCACAGGGGAGUUGUGU-3' (SEQ ID NO: 52)
3'-AGGAGUGUCCCCUCAACACA-5' (SEQ ID NO: 53)
[0278] This has the effect of producing a more balanced
representation of weak (A:U) and strong (G:C) base pairs at the
ends of the duplex.
[0279] Alternatively, a duplex for which the TNFa-targeting strand
is mutated but still capable of binding to the target and the
corresponding strand is changed to match could be: TABLE-US-00013
5'-CCUCACAGGGGAGUUGUGC-3' (SEQ ID NO: 54) 3'-GGAGUGUCCCCUCAACACG-5'
(SEQ ID NO: 55)
[0280] In this situation, the first 5 base pairs of the duplex are
equally balanced at the two ends, without appreciably compromising
binding to the target as shown below (note wobble-base pair with
TNFa-targeting strand). TABLE-US-00014 HCV 5'
GGCGACACUCCACCAUGAAUCACUCCCCUGUGAGGA 3' (SEQ ID NO: 56) Guide#1 3'
CGUGUUUGAGGGGACACUCC 5' (SEQ ID NO: 54) mfe: -34.7 kcal/mol TNFa 5'
GCCUCUGCUCCCCAGGGGAGUUGUGUC 3' (SEQ ID NO: 51) Guide#2 3'
GGAGUGUCCCCUCAACACG 5' (SEQ ID NO: 55) mfe: -35.3 kcal/mol
[0281] If required, overhangs can be added. It may be beneficial to
make those complementary to the intended target so as to enhanced
improved binding of the tail region. The added bases may be
selected so as to provide predicted binding to a specified further
region in the target RNA. For example, in the above in silico
binding result a large bulge is predicted to be formed from the
binding of the first guide strand to the HCV target RNA. The choice
of overhangs could be guided by the desire to reduce the length of
that bulge. Other alternatives are to add bases complementary to
the target to provide an extension of the binding indicated in
silico. So, as an example, the duplex generated above could be
further extended using the information from the in silico
hybridization to further define the bases required. TABLE-US-00015
5'-CCUCACAGGGGAGUUGUGCCC-3' (SEQ ID NO: 57)
3'-UCGGAGUGUCCCCUCAACACG-5' (SEQ ID NO: 58)
[0282] Alternatively, the sequence could be empirically extended by
adding UU: TABLE-US-00016 5'-CCUCACAGGGGAGUUGUGCUU-3' (SEQ ID NO:
59) 3'-UUGGAGUGUCCCCUCAACACG-5' (SEQ ID NO: 60)
[0283] It will be appreciated by one skilled in the art that
synthetic multitargeting interfering RNA duplexes will comprise the
seed and its corresponding complement (5' CAGGGGAGU 3'). (SEQ ID
NO: 46)
[0284] Other seeds perhaps worthy of further investigation are 5'
CGCCTGGAGCCCT 3' (SEQ ID NO: 61) and 5' CTCCTCGGCCAGC 3'. (SEQ ID
NO: 62)
[0285] It will be appreciated by one skilled in the art that
synthetic multitargeting interfering RNA duplexes will comprise the
seed and its corresponding complement (5' AGGGCUCCAGGCG 3' (SEQ ID
NO: 63) or 5' GCUGGCCGAGGAG 3'). (SEQ ID NO: 64)
EXAMPLE 5
Modifications to Improve Strand Loading
[0286] A likely explanation for the decreased activity of
blunt-ended CODEMIRs is that RISC loading is impaired in the
absence of 3' overhangs. We have investigated the use of a single
blunt-end to prevent loading of one strand of a CODEMIR; a
technique that is potentially useful for promoting loading of the
guide strand. For this study, we designed a variant of CODEMIR-17,
which has a strong preference for loading of the ICAM targeting
guide strand. The variant CODEMIR-103 (Table 5-1) was designed to
include a blunt-end at the 5' end of the ICAM-1 guide strand. This
CODEMIR demonstrated increased VEGF suppressive activity, and
decreased ICAM-1 suppressive activity (FIG. 3), which is consistent
with altered strand loading.
[0287] In this example, ARPE-19 cells were transfected with 40 nM
duplex RNA and VEGF (ELISA) or ICAM (FACS) expression was assayed
48 hours post-transfection. Each bar in FIG. 3 represents the mean
of triplicate samples. Error bars indicate standard deviation of
the mean. TABLE-US-00017 TABLE 5-1 Sequences of CODEMIR-17 and its
single-blunt-ended variant CODEMIR-103. Duplex (top strand 5' to
3'; bottom strand 3' to 5') mRNA binding (RNA hybrid) CODEMIR-17
ICAM Guide VEGF UCGAGUGACAGUCACUAGCUC (SEQ ID NO: 7) 5' UGAUCG
GUGACAGUCACUAGCU 3' (SEQ ID NO: 9) CUAGCUCACUGUCAGUGAUCG (SEQ ID
NO: 8) 3' CUAGCUCACUGUCAGUGAUCG 5' (SEQ ID NO: 8) VEGF Guide ICAM
5' GGGGAAUCAGUGACUGUCACUCGAG 3' (SEQ ID NO: 10) 3'
CUCGAUCACUGACAGUGAGCU 5' (SEQ ID NO: 7) CODEMIR-103 ICAM Guide VEGF
GAUCGAGUGACAGUCACUAGCUC (SEQ ID NO: 65) 5' UGAUCG GUGACAGUCACUAGCU
3' (SEQ ID NO: 9) CUAGCUCACUGUCAGUGAUCG (SEQ ID NO: 8) 3'
CUAGCUCACUGUCAGUGAUCG 5' (SEQ ID NO: 8) VEGF Guide ICAM 5'
GGGGAAUCAGUGACUGUCACUCGAGA 3' (SEQ ID NO: 66) 3'
CUCGAUCACUGACAGUGAGCUAG 5' (SEQ ID NO: 65)
EXAMPLE 6
Activity of CODEMIRs and VIROMIRs In Vivo
[0288] The activity of CODEMIRs and other multitargeting
interfering RNA of the invention could be tested in various
preclinical models known to those skilled in the art. As a
non-limiting example, CODEMIRs-26-28 could be tested in a
retinopathy of prematurity model. This model is well known to those
working in the field of ocular angiogenesis and is used extensively
as one of several models for the development of drugs active
against the diseases of interest (AMD, diabetic retinopathy etc).
The study could comprise of a suitable number of mouse or rat
neonate pups equally divided into treatment groups. The treatment
groups could include negative controls such as vehicle, irrelevant
or scrambled sequence controls plus a number of multitargeting
interfering RNA. One could also consider including siRNA to VEGF as
known comparators.
[0289] In this model, beginning on Day 1 of life, litters are
exposed to cycles of hyperoxia followed by several days of room
air. The injections could be performed on the last day of cycling,
prior to the 4 day normoxia period. Several days later, animals
could be injected with FITC-dextran and sacrificed. Fluorescence
images of the retinal flat mounts could used to estimate the extent
of neoangiogenesis in each animal. In addition, measurement of the
production of the target RNA molecules or their encoded proteins
(in this case, VEGF and ICAM) could be made by analysis of
homogenized samples or, alternatively, with in situ
hybridization.
[0290] As a further non-limiting example, CODEMIRs could
alternatively be evaluated in vivo for inhibition of
disease-related angiogenesis using the laser-induced Choroidal
Neovascularization (CNV) model in rats or primates. In this model,
animals under general anaesthesia have their pupils dilated and
retina photographed. Choroidal neovascularisation (CNV) is induced
by krypton laser photocoagulation. This is performed using laser
irradiation to either the left or alternatively, the right eye of
each animal from all treatment groups through a slit lamp. A total
of 6-11 laser burns are generally applied to each eye surrounding
the optic nerve at the posterior pole.
[0291] At a suitable time following laser injury, the
multitargeting interfering RNA are injected into the affected eyes.
The suitable time can be the day following laser induction, or for
an assessment against established CNV, the injections can be
performed several days or weeks following injury. Intravitreal
injections of the oligonucleotides are performed by inserting a 30-
or 32-gauge needle into the vitreous. Insertion and infusion can be
performed and directly viewed through an operating microscope. Care
is taken not to injure the lens or the retina. Ideally, the test
compounds are placed in the superior and peripheral vitreous
cavity. Periodically after treatment, the neoangiogenesis is
evaluated by either imaging and/or direct sampling (eg histology,
immunohistochemistry). In all cases, the assessment of CNV is best
performed by a skilled operator blinded to the actual treatment to
ensure a lack of bias in the recording of the information.
[0292] An example of a direct imaging method is Colour Fundus
Photography (CFP). Again, under anaesthesia as described above, the
pupils are dilated. The fundus is then photographed with a camera
using the appropriate film.
[0293] Alternatively, or preferably in addition to CFP, fluorescein
angiography is used to image the vessels and areas of vascular
leakage in the retina. This is performed on all of the animals
following the intraperitoneal or intravenous injection of sodium
fluorescein. The retinal vasculature is then photographed using the
same camera as used for CFP but with a barrier filter for
fluorescein angiography added. Single photographs can be taken at
0.5-1 minute intervals immediately after the administration of
sodium fluorescein. The extent of fluorescein leakage is scored by
a trained operator. The mean severity scores from each of the time
points are compared by a suitable statistical analysis and
differences considered significant at p<0.05. In addition, the
frequency of each lesion score is counted, tabulated and
represented graphically.
[0294] Alternatively, or in addition, rats can be euthanased at
selected time points following treatment (for example 7, 14 and 28
days post injection) and eyes examined by conventional
histopathology. A reduction in the number and severity of lesions
is expected to be seen with samples treated by active
oligonucleotides of the invention.
[0295] Other non-limiting examples including testing the
multitargeting interfering RNA of the invention in other
preclinical models such as those that are well known to those
skilled in the art. A non-exhaustive list includes pulmonary
fibrosis (bleomycin induced), paw inflammation (carrageen), joint
arthritis, diabetes, viral infection, tumour xenografts etc.
[0296] While the foregoing specification teaches the principles of
the present invention, with examples provided for the purpose of
illustration, it will be understood that the practice of the
invention encompasses all of the usual variations, adaptations
and/or modifications as come within the scope of the following
claims and their equivalents. All references are hereby
incorporated into this application in their entirety.
Sequence CWU 1
1
67 1 12 DNA artificial oligonucleotide 1 agtgactgtc ac 12 2 12 RNA
artificial complementary oligonucleotide 2 gugacaguca cu 12 3 21
RNA artificial ICAM guide oligonucleotide 3 cgagugacag ucacuagcuc c
21 4 21 RNA artificial VEGF Guide Oligonucleotide 4 agcuagugac
ugucacucga u 21 5 22 RNA artificial VEGF binding oligonucleotide 5
gaucggugac agucacuagc uu 22 6 25 RNA artificial ICAM binding
oligonucleotide 6 cggggaauca gugacuguca cucga 25 7 21 RNA
artificial ICAM guide oligonucleotide 7 ucgagugaca gucacuagcu c 21
8 21 RNA artificial VEGF Guide oligonucleotide 8 gcuagugacu
gucacucgau c 21 9 22 RNA artificial VEGF binding oligonucleotide 9
ugaucgguga cagucacuag cu 22 10 25 RNA artificial ICAM binding
oligonucleotide 10 ggggaaucag ugacugucac ucgag 25 11 22 RNA
artificial ICAM guide oligonucleotide 11 ucgagugaca gucacuagcu cc
22 12 22 RNA artificial VEGF Binding oligonucleotide 12 agcuagugac
ugucacucga uc 22 13 26 RNA artificial ICAM binding oligonucleotide
13 cggggaauca gugacuguca cucgag 26 14 21 RNA artificial VEGF Guide
oligonucleotide 14 agcuagugac ugucacuuga u 21 15 21 RNA artificial
ICAM guide oligonucleotide 15 ucgagugaca gucacuaguu c 21 16 21 RNA
artificial ICAM guide oligonucleotide 16 cgagugacag ucacugauuc c 21
17 21 RNA artificial VEGF guide oligonucleotide 17 agcuagugac
ugucaccgau c 21 18 23 RNA artificial ICAM binding oligonucleotide
18 gggaaucagu gacugucacu cga 23 19 13 RNA artificial
oligonucleotide in glucose-g-phosphatase and Inppl1 19 cugccucgcc
cag 13 20 12 RNA artificial oligonucleotide in
glucose-6-phosphatase and Inppl1 20 cuccacaucc ac 12 21 13 RNA
artificial oligonucleotide complementary to SEQ ID NO19 21
cugggcgagg cag 13 22 12 RNA artificial oligonucleotide
complementary to SEQ ID NO20 22 guggaugugg ag 12 23 39 RNA
artificial Gluc6p oligonucleotide 23 gugucauccc cuacugccuc
gcccaggucc ugggccagc 39 24 39 RNA artificial Inppl1 complement
oligonucleotide 24 caggcacuca ugccugccuc gcccagcccg cuggcccgc 39 25
21 RNA artificial Inppl1 targeting strand 25 augccugccu cgcccagguc
c 21 26 21 RNA artificial Gluc6p targeting strand 26 ggaccugggc
gaggcaggca u 21 27 41 RNA artificial Gluc6p oligonucleotide 27
gauucuucca cuggcuccac auccacccca cuggaucuuc a 41 28 41 RNA
artificial Inppl1 complement oligonucleotide 28 accagccgcc
cacccuccac auccacgcuc agcgugaacu u 41 29 21 RNA artificial Inppl1
targeting strand 29 cacccuccac auccacccca c 21 30 21 RNA artificial
Gluc6p targeting strand 30 guggggugga uguggagggu g 21 31 83 DNA
artificial HIV seed oligonucleotide 31 tctaattcca ataattcttg
ttcattcttt tcttgctggt tttgcgattc ttcaattaag 60 gagtgtatta
agcttgtgta att 83 32 83 DNA artificial HIV complementary
oligonucleotide 32 ctttgagcca attcccatac attattgtgc cccggctggt
tttgcgattc taaaatgtaa 60 taataagacg ttcaatggaa cag 83 33 17 DNA
artificial HIV seed sequence 33 gctggttttg cgattct 17 34 17 DNA
artificial HIV seed complementary sequence 34 agaatcgcaa aaccagc 17
35 15 DNA artificial modified seed sequence 35 tggttttgcg attct 15
36 15 DNA artificial modified complementary sequence 36 agaatcgcaa
aacca 15 37 21 RNA artificial viromir duplex sequence 37 ugcugguuuu
gcgauucuaa a 21 38 21 RNA artificial Viromir targetting sequence 38
uagaaucgca aaaccagcaa g 21 39 66 DNA artificial shRNA Viromir 39
gatccgcttg ctggttttgc gattctaact cgagatagaa tcgcaaaacc agcaagtttt
60 ttggaa 66 40 66 DNA artificial shRNA complementary strand 40
agctttccaa aaaacttgct ggttttgcga ttctatctcg agttagaatc gcaaaaccag
60 caagcg 66 41 9 DNA artificial seed sequence in HCV and TNFalpha
41 actcccctg 9 42 15 DNA artificial extended seed sequence 42
actcccctgt gagga 15 43 15 DNA artificial extended seed sequence 43
actcccctga cgccg 15 44 66 DNA artificial HCV with seed sequence 44
tgatgggggc gacactccac catgaatcac tcccctgtga ggaactactg tcttcacgca
60 gaaagc 66 45 66 DNA artificial TNFalpha seed sequence 45
gacagtgagt aacgactcgg agacgagggg tcccctcaac acagacatta gcgggatgat
60 aagtca 66 46 9 RNA artificial HCV seed sequence 46 caggggagu 9
47 14 RNA artificial HCV seed sequence 47 ccucacaggg gagu 14 48 19
RNA artificial TNFalpha sequence 48 ccucacaggg gaguugugu 19 49 19
RNA artificial TNFalpha complement sequence 49 acacaacucc ccugugagg
19 50 33 RNA artificial guide strand sequence 50 gacacuccac
caugaaucac uccccuguga gga 33 51 27 RNA artificial TNFalpha guide
sequence 51 gccucugcuc cccaggggag uuguguc 27 52 20 RNA artificial
HCV extension sequence 52 uccucacagg ggaguugugu 20 53 20 RNA
artificial HCV guide sequence 53 aggagugucc ccucaacaca 20 54 19 RNA
artificial alternative TNFalpha sequence 54 ccucacaggg gaguugugc 19
55 19 RNA artificial alternative TNFalpha complementary strand
sequence 55 gcacaacucc ccugugagg 19 56 36 RNA artificial HCV strand
sequence 56 ggcgacacuc caccaugaau cacuccccug ugagga 36 57 21 RNA
artificial HCV sequence 57 ccucacaggg gaguugugcc c 21 58 21 RNA
artificial HCV complementary strand sequence 58 gcacaacucc
ccugugaggc u 21 59 21 RNA artificial extended sequence of SEQ ID
NO57 59 ccucacaggg gaguugugcu u 21 60 21 RNA artificial extended
SEQ ID NO58 sequence 60 gcacaacucc ccugugaggu u 21 61 13 DNA
artificial alternative seed sequence 61 cgcctggagc cct 13 62 13 DNA
artificial alternative seed sequence 62 ctcctcggcc agc 13 63 13 RNA
artificial seed of multitargeting interfering RNA duplex 63
agggcuccag gcg 13 64 13 RNA artificial seed of synthetic
multitargeting interfering RNA duplex complement 64 gcuggccgag gag
13 65 23 RNA artificial ICAM mRNA binding (RNA hybrid) 65
gaucgaguga cagucacuag cuc 23 66 26 RNA artificial ICAM mRNA binding
sequence 66 ggggaaucag ugacugucac ucgaga 26 67 23 RNA artificial
VEGF binding sequence 67 ugaucgguga cagucacuag cuu 23
* * * * *